hGC-1, a gene encoding a member of the olfactomedin-related protein family

ABSTRACT

An isolated acid having the sequence of a) SEQ ID NO: 1; b) the sequence of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 3; d) a sequence complementary to any of a), b), or c); or e) a sequence of at least 10 contiguous nucleotides specific for any of a)-d). The invention relates to the identification and characterization of a hitherto unidentified human gene, hGC-1. The protein encoded by hGC-1 appears to be a member of the olfactomedin-related proteins. The invention relates generally to the gene (hGC-1), nucleic acids, cDNA, vectors, polypeptides, protein, antibodies, cells, transgenic animal, and other compositions related to hGC-1. Additionally, primers are provided for identifying hGC-1. The invention further relates to methods of using these compositions, such as diagnosis and treatment of various cancers, and kits comprising these compositions.

This application claims priority to U.S. application Ser. No.60/338,759, filed Dec. 7, 2001, which is hereby incorporated in itsentirety by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the identification andcharacterization of a hitherto unidentified human gene, hGC-1. Theinvention relates generally to the gene (hGC-1), nucleotide sequences,vectors, polypeptides, antibodies, and other compositions related tohGC-1. Additionally, primers are provided for identifying hGC-1. Theinvention further relates to methods of using these compositions, suchas diagnosis and treatment of various cancers, and kits comprising thesecompositions.

2. Background

During the process of hematopoiesis, pluripotent hematopoietic stemcells become committed to one lineage and eventually differentiate intofunctional, morphologically distinct end-stage cells (Akashi, K.,Traver, D., Miyamoto, T. & Weissman, I. L. (2000) Nature 404, 193-7). Inthe bone marrow, pluripotent stem cells differentiate into either thelymphoid stem cell line, where they are further induced to differentiateinto B- or T-derived lymphocytes, or the myeloid stem cell (CFU-GEMM)line, where they are further induced to become erythrocytes,granulocytes (neutrophils, eosinophils, or basophils), macrophages, ormegakaryocytes (platelets).

Proliferation and differentiation of blood cells in the bone marrow areregulated by hematopoietic factors. Hematopoietic factors that arecontinuously produced include erythropoietin (EPO), granulocytecolony-stimulating factor (G-CSF) and thrombopoietin (TPO). EPO, G-CSFand TPO bind to their corresponding receptors, thereby inducing tyrosinephosphorylation of a number of cellular proteins and activating specificintracellular signaling cascades, including the signal transducer andactivator of transcription (STAT) and mitogen-activated protein kinase(MAPK) pathways (Tidow, N. & Welte, K. (1997) Curr Opin Hematol 4,171-5; Wojchowski, D. M., Gregory, R. C., Miller, C. P., Pandit, A. K. &Pircher, T. J. (1999) Exp Cell Res 253, 143-56; Alexander, W. S. (1999)Growth Factors 17, 13-24). Differentiation in response to hematopoieticfactor signaling is accompanied by coordinate expression of specificgenes. However, knowledge about the differences in the signaltransduction pathways and gene expression profiles stimulated by thesethree hematopoietic factors remains limited.

Identification of elements that regulate hematopoietic differentiationand of the genes expressed in response to such regulators is an activearea of research. For example, genetic studies in mice have revealedcritical lineage-specific roles for transcriptional regulators such asC/EBP (Zhang, D. E., Zhang, P., Wang, N. D., Hetherington, C. J.,Darlington, G. J. & Tenen, D. G. (1997) Proc Natl Acad Sci USA 94,569-74), GATA-1 (Pevny, L., Lin, C. S., D'Agati, V., Simon, M. C.,Orkin, S. H. & Costantini, F. (1995) Development 121, 163-72), PU.1(Scott, E. W., Simon, M. C., Anastasi, J. & Singh, H. (1994) Science265, 1573-7), and many others (Ness, S. A. & Engel, J. D. (1994) CurrOpin Genet Dev 4, 718-24). Elucidation of the genetic alterationsunderlying certain leukemias, including chromosomal translocations andmore subtle mutations, has revealed the hematopoietic functions ofproteins such as PLZF and AML-1 (Shivdasani, R. A. & Orkin, S. H. (1996)Blood 87, 4025-39).

A mRNA differential display approach was used to examine potentiallynovel genes associated with hematopoietic lineage commitment and toexplore new lineage-specific markers for monitoring lineagedifferentiation. One of these cDNA fragments derived fromdifferentiation pathways of various lineages was cloned and sequenced.hGC-1 (human G-CSF-stimulated clone-1), is selectively expressed innormal human myeloid lineage cells and is a marker for various cancers.

The following describes the identification and characterization of thehGC-1 gene, its induction properties in hematopoietic cells, andpotentially important aspects of its corresponding extracellularprotein/glycoprotein structure. Additionally, related compositions andmethods for using those compositions are described.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, relates to the identificationand characterization of a hitherto unidentified human gene, hGC-1. Theprotein encoded by hGC-1 appears to be a member of theolfactomedin-related proteins. The invention relates generally to thegene (hGC-1), nucleic acids, cDNA, vectors, polypeptides, protein,antibodies, cells, transgenic animals, and other compositions related tohGC-1. Additionally, primers and probes are provided for identifyinghGC-1. The invention further relates to methods of using thesecompositions, such as diagnosis and treatment of various cancers, andkits comprising these compositions.

The findings of the invention indicate that hGC-1 is primarily expressedas an extracellular olfactomedin-related glycoprotein during normalmyeloid-specific lineage differentiation, suggesting the possibility ofa matrix-related function for hGC-1 in differentiation. The inventionestablishes a link between hGC-1 and various cancers, including myeloma,B-cell leukemia, and prostate cancer.

Advantages of the invention will be set forth in part in the descriptionwhich follows, and in part will be obvious from the description, or maybe learned by practice of the invention. The advantages of the inventionwill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various aspects of the inventionand together with the description, serve to explain the principles ofthe invention.

FIG. 1. Overview of the two-phase liquid culture system. Phase I allowsfor the synchronization, proliferation and differentiation of early(myeloid) stem cell progenitors. Phase II permits lineage-specificproliferation and differentiation under the influence of G-CSF, EPO andTPO. The cells are further enriched by cell sorting (FACS) withlineage-specific markers.

FIG. 2. (A) Differential display of PCR products from total RNAs ofglycophorin A+ (erythroid), CD13+ (myeloid) and CD61+ (megakaryocytic)cells. [α-³³P]-dATP-labeled PCR products amplified with 5′-AP primersand 3′-oligo(dT) primers were electrophoresed through a 6% denaturingpolyacrylamide gel and exposed to x-ray film for 24 hours. Thedifferentially displayed hGC-1 fragment is indicated by arrows. (B)Deduced cDNA and protein sequence of hGC-1 (SEQ ID NO:4). Nucleotidescomposing the region identical to the original DD-PCR fragment areunderlined with a single solid line. The N-terminal amino acid of maturehGC-1 is aspartic acid, indicated by an arrow. It is preceded by a20-amino acid signal peptide, indicated by the boxed area, whichcontains the initiating methionine. hGC-1 protein contains six potentialN-linked glycosylation sites, indicated by arrowheads. Thepolyadenylation motifs are further underlined with curved dashed lines.The star indicates the termination codon.

FIG. 3. Chromosomal localization and gene structure of hGC-1. (A) Thegene is shown schematically with its five exons (boxes) and introns(lines). Transcription initiation sites are indicated by arrows. (B)FISH using BAC DNA containing the hGC-1 sequence as a probe. The probewas labeled with digoxigenin and visualized by FITC (red). Fluorescencesignals were detected at chromosome 13q14.3 as indicated by arrows. (C)Idiogram of human chromosome 13, illustrating the location of the hGC-1gene on chromosome band 13q14.3.

FIG. 4. Northern blot hybridization analysis of hGC-1 expression.Northern blots were hybridized under stringent conditions with uniformlylabeled full-length hGC-1 probes, as described in Methods and Materials.Numbers on the right indicate positions of hGC-1 bands. (A) hGC-1expression in Phase II, day 5, FACS-selected hematopoietic cells and infour leukemia cell lines. (B) Expression of hGC-1 in multiple humantissues was determined by hybridization cDNA probes to Multiple TissueNorthern (MTN™) Blots (Clontech).

FIG. 5. (A and B) Induction of hGC-1 expression in a two-phase liquidculture system and in the HL-60 cell line. (A) Wright-Giemsa-stainedcytocentrifuge preparations of cells from two-phase liquid culturesinduced to differentiate toward the erythroid, myeloid, andmegakaryocytic lineages. Representative images from cultures at 1, 5 and11 days are shown. (B) EtBr-stained PAGE of the RT-PCR products of thehGC-1 and β-actin mRNAs from two-phase cell cultures collected on daysindicated. (C and D) Induction of hGC-1 expression duringdifferentiation of HL-60 cells toward granulocytes and monocytes. (C)Wright-Giemsa-stained cytocentrifuge preparations of HL-60 cells inducedto differentiate toward granulocytes and monocytes. Representativeimages from cultures at 1, 2, 3, 5 and 7 days are shown. (D)EtBr-stained PAGE of RT-PCR products of the hGC-1 and β-actin mRNAs fromHL-60 cells collected on the days indicated.

FIG. 6. (A) Chou-Fasman analysis. Predicted secondary structures ofolfactomedin and hGC-1 protein: α-helices are shown with a sine wave,β-sheets with a sharp saw-tooth wave, turns with 180 degree turns, andcoils with a dull saw-tooth wave. Red indicates KD hydrophilicity≧1.3;purple indicates KD hydrophobicity≧1.3. (B) hGC-1 sequence homology toolfactomedin-related proteins: comparison of olfactomedin from bullfrogolfactory tissue, hGC-1, noelin-2, TIGR and latrophilin-1. hGC-1 showedsignificant homology to the mucus glycoprotein olfactomedin. A stretchof 233 amino acids in the carboxyl terminus of hGC-1 is highly conservedamong all these proteins.

FIG. 7. (A) In vitro transcription-coupled translation andN-glycosylation analysis of hGC-1 protein. In vitro translation productswith and without CPMM treatment were analyzed by 12% SDS-PAGE. Lane 1,pCRII-hGC-1; lane 2, pcDNA-E-hGC-1-his/V5; pcDNA-E-hGC-1-his/V5+CPMM.(B) Western blot analysis and N-glycanase treatment of hGC-1. Celllysate proteins were prepared from either parental 293 cells or 293cells transfected with pcDNA-E-hGC-1-his/V5, fractionated by 12%SDS-PAGE, and blotted onto Hybond NC membranes. hGC-1 was visualized byan anti-V5 mab. The dilution of the antibody is shown. Parental 293 celllysates were used as controls (lanes 1 and 2). Cells were incubated ineither the presence (+) or absence (−) of the N-glycanase PNGase.Molecular mass markers are shown in the left margin.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to the specific methods of making and usingthem, since they may, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

Definitions and Use of Terms

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a nucleic acid” includes mixtures of nucleic acids,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

By an “effective amount” of a compound as provided herein is meant asufficient amount of the compound to provide the desired effect. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofdisease (or underlying genetic defect) that is being treated, theparticular compound used, its mode of administration, and the like.Thus, it is not possible to specify an exact “effective amount.”However, an appropriate “effective amount” may be determined by one ofordinary skill in the art using only routine experimentation.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected compound withoutcausing any undesirable biological effects or interacting in aundesirable manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier maydepend on the method of administration and the particular patient.

“hGC-1” refers to the gene or nucleic acid. A hGC-1 gene or nucleic acidrefers to any gene or nucleic acid identified with or derived from awild-type hGC-1 gene. For example, a mutant hGC-1 gene is a form of ahGC-1 gene.

The term “gene” as used herein means a unit of heredity that occupies aspecific locus on a chromosome as well as any sequences associated withthe expression of that nucleic acid. For example, the gene includes anyintrons normally present within the coding region as well as regionspreceding and following the coding region. Examples of these non-codingregions include, but are not limited to transcription terminationregions, promoter regions, enhancer regions and modulation regions.Since the genomic location of the hGC-1 gene is provided herein, thepresent invention includes any examples of the hGC-1 gene that occur atthat locus.

As used herein, the term “nucleic acid” refers to single-or multiplestranded molecules which may be DNA or RNA, or any combination thereof,including modifications to those nucleic acids. The nucleic acid mayrepresent a coding strand or its complement, or any combination thereof.Nucleic acids may be identical in sequence to the sequences which arenaturally occurring for any of the novel genes discussed herein, or theymay include alternative codons which encode the same amino acid as thatwhich is found in the naturally-occurring sequence. These nucleic acidscan also be modified from their typical structure. Such modificationsinclude, but are not limited to, methylated nucleic acids, thesubstitution of a non-bridging oxygen on the phosphate residue witheither a sulfur (yielding phosphorothioate deoxynucleotides), selenium(yielding phosphorselenoate deoxynucleotides), or methyl groups(yielding methylphosphonate deoxynucleotides).

As used herein, the term “specific” refers to having a unique relationto. For example, “species specific” refers to an amino acid or nucleicacid sequence that is found only in a particular species. In anotherexample, “protein/nucleic acid-specific” in the context of a fragmentrefers to an amino acid or nucleic acid fragment of the referencedprotein or nucleic acid that is found only in the referenced protein ornucleic acid.

As used herein, the term “isolated” refers to a nucleic acid separatedor significantly free from at least some of the other components of thenaturally occurring organism, for example, the cell structuralcomponents commonly found associated with nucleic acids in a cellularenvironment and/or other nucleic acids. The isolation of the nativenucleic acids can be accomplished, for example, by techniques such ascell lysis followed by phenol plus chloroform extraction, followed byethanol precipitation of the nucleic acids. The nucleic acids of thisinvention can be isolated from cells according to any of many methodswell known in the art.

By “isolated nucleic acid” or “purified nucleic acid” is meant DNA thatis free of some or all of the genes that, in the naturally-occurringgenome of the organism from which the DNA of the invention is derived,flank the gene. The term therefore includes, for example, a recombinantDNA which is incorporated into a vector, such as an autonomouslyreplicating plasmid or virus; or incorporated into the genomic DNA of aprokaryote or eukaryote (e.g., a transgene); or which exists as aseparate molecule (for example, a cDNA or a genomic or cDNA fragmentproduced by PCR, restriction endonuclease digestion, or chemical or invitro synthesis). It also includes a recombinant DNA which is part of ahybrid gene encoding additional polypeptide sequence. The term “isolatednucleic acid” also refers to RNA, e.g., an mRNA molecule that is encodedby an isolated DNA molecule, or that is chemically synthesized, or thatis separated or substantially free from at least some cellularcomponents, for example, other types of RNA molecules or polypeptidemolecules.

As used herein, the terms “genomic variant” and “allelic variant” mean asimilar gene in another organism of the same species. For example, anucleic acid from one member of a species can encode a particular hGC-1protein while another member of the same species has a different nucleicacid which encodes that same hGC-1 protein. A similar gene in anotherspecies is defined herein as a “homolog” of a hGC-1 protein. Forexample, a hGC-1 protein from one species can be different than a hGC-1protein from another species, yet both may have the same function as theexemplified hGC-1 protein.

By “hGC-1 biological activity” is meant any physiological functionattributable to a hGC-1 polypeptide molecule, including signaltransduction. hGC-1 biological activity, as referred to herein, isrelative to that of the normal hGC-1 polypeptide molecule. It may bedesirable to increase or decrease hGC-1 biological activity.

Mechanisms by which a compound may increase hGC-1 biological activityinclude, but are not limited to, mimicry of endogenous hGC-1 polypeptideactivity; stimulation of the activity of a less active or inactiveversion (for example, a mutant) of the hGC-1 polypeptide; or increasingthe amount of hGC-1 polypeptide in a cell (for example, by stimulatinghGC-1 transcription and/or translation or by inhibiting hGC-1 mRNA orpolypeptide degradation).

hGC-1 biological activity in a sample, such as a cell, tissue, oranimal, may be indirectly measured by measuring the relative amount ofhGC-1 mRNA (for example, by reverse transcription-polymerase chainreaction (RT-PCR) amplification, ribonuclease protection assay orNorthern hybridization); the level of hGC-1 polypeptide (for example, byELISA or Western blotting); or the activity of a reporter gene under thetranscriptional regulation of a hGC-1 transcriptional regulatory region(by reporter gene assay, for example, employing beta-galactosidase,chloramphenicol acetyltransferase (CAT), luciferase, or greenfluorescent protein, as is well known in the art). For example, acompound that increases the amount of wild-type hGC-1 polypeptide (orany other version of the polypeptide that maintains at least someactivity) in a cell is a compound that increases biological activity ofhGC-1.

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably and as used herein refer to more than one amino acidjoined by a peptide bond.

By “hGC-1 polypeptide” is meant a polypeptide that has, or is relatedto, the amino acid sequence of SEQ ID NO:4. A hGC-1 polypeptide containsan amino acid sequence that bears at least 70% sequence identity, to theamino acid sequence of SEQ ID NO:4.

“Isolated” or “purified” is meant to encompass items, e.g., peptides,which are not naturally occurring, in that they are isolated, purified,synthesized, or otherwise manipulated by man. The isolated or purifiedpeptides, polypeptide, proteins or nucleic acids need not behomogeneous, but must be sufficiently separated from the othercomponents in their naturally environment to allow them to be used forclinical diagnosis or treatment, or for scientific research. By“isolated polypeptide” or “purified polypeptide” is meant a polypeptide(or a fragment thereof) that is substantially free from the materialswith which the polypeptide is normally associated in nature. Thepolypeptides of the invention, or fragments thereof, can be obtained,for example, by extraction from a natural source (for example, amammalian cell), by expression of a recombinant nucleic acid encodingthe polypeptide (for example, in a cell or in a cell-free translationsystem), or by chemically synthesizing the polypeptide. In addition,polypeptide fragments may be obtained by any of these methods, or bycleaving full length polypeptides.

By “wild-type hGC-1 polypeptide” is meant a hGC-1 polypeptide that hasnormal biological activity, e.g., is produced by a normal subject notsuffering from or predisposed to diseases affected by the polypeptide.An example of a wild-type hGC-1 polypeptide is the amino acid sequenceof SEQ ID NO:4.

By “wild-type hGC-1 nucleic acid” is meant a nucleic acid that encodes awild-type hGC-1 polypeptide. Examples of a wild-type hGC-1 nucleic acidsinclude SEQ ID NO:2 and the hGC-1 ORF shown in FIG. 2. Other wild-typehGC-1 nucleic acids include those containing introns, such as genomichGC-1 nucleic acid, SEQ ID NO:1.

By “polymorphic variant of an hGC-1 polypeptide” is meant an hGC-1polypeptide containing an amino acid change, relative to wild type, thatdoes not cause disease. Such polymorphic amino acid variations in hGC-1are seen in both patients and in normal individuals. However, apolymorphic variant, while not the underlying cause of a disease orclinical condition, may subtly increase or decrease hGC-1 biologicalactivity such that hGC-1 is either more efficient or less efficient thanthat performed by a wild type hGC-1 polypeptide molecule.

By “mutant hGC-1 polypeptide” is meant an hGC-1 polypeptide thatprematurely terminates (i.e., is not full length) or that contains anamino acid substitution such that the polypeptide displays lessbiological activity than the wild type hGC-1 polypeptide, e.g., becauseit is less stable than the wild type polypeptide (and is thus degradedmore rapidly). Examples of mutant hGC-1 polypeptides are those encodedby the genes of patients suffering from cancer or hyperplasia, asdescribed herein.

By “mutated hGC-1 nucleic acid” is meant a nucleic acid that encodes amutant hGC-1 polypeptide.

By “transfected” or “transformed” is meant an exogenous gene physicallyintroduced into a cell or culture of cells, such that the new nucleicacid is detectable, can be expressed as an RNA or protein, or can bepassed on to successive generations.

By “increased susceptibility for developing cancer” is meant a subjectwho has a greater than normal chance of developing cancer, compared tothe general population. Such subjects include, for example, a subjectthat harbors a mutation in a hGC-1 gene such that biological activity ofhGC-1 polypeptide is altered.

By “test compound” is meant a molecule, be it naturally-occurring orartificially derived, that is surveyed for its ability to modulate hGC-1activity. Test compounds may include, for example, peptides,polypeptides, synthesized organic molecules, naturally occurring organicmolecules, nucleic acid molecules, and components thereof.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; or a solution containing one or moremolecules derived from a cell or cellular material (e.g., a polypeptideor nucleic acid), which is assayed as described herein. A sample mayalso be any body fluid or excretion (for example, but not limited to,blood, urine, stool, saliva, tears, bile) that contains cells or cellcomponents.

By “modulate” is meant to alter, by increase or decrease.

By “normal subject” is meant an individual who does not have anincreased susceptibility for developing cancer.

The “subject” or “patient” of this method can be any animal. The animalof the present invention may be a human. In addition, non-human animalswhich can be treated by the methods of this invention can include, butare not limited to, cats, dogs, birds, horses, cows, goats, sheep,guinea pigs, hamsters, gerbils and rabbits.

By a “transgene” is meant a nucleic acid sequence that is inserted byartifice into a cell and becomes a part of the genome of that cell andits progeny. Such a transgene may be (but is not necessarily) partly orentirely heterologous (for example, derived from a different species) tothe cell.

By “transgenic animal” an animal comprising a transgene as describedabove. Transgenic animals are made by techniques that are well known inthe art.

By “knockout mutation” is meant an alteration in the nucleic acidsequence that reduces the biological activity of the polypeptidenormally encoded therefrom by at least 80% relative to the unmutatedgene. The mutation may, without limitation, be an insertion, deletion,frameshift, or missense mutation. A “knockout animal,” for example, aknockout mouse, is an animal containing a knockout mutation. Theknockout animal may be heterozygous or homozygous for the knockoutmutation. Such knockout animals are generated by techniques that arewell known in the art. A form of knockout mutation is one where thebiological activity of the hGC-1 polypeptide is not completelyeliminated.

By “treat” is meant to administer a compound or molecule of theinvention to a subject, such as a human or other mammal (for example, ananimal model), that has an increased susceptibility for developingcancer, or that has cancer, in order to prevent or delay a worsening ofthe effects of the disease or condition, or to partially or fullyreverse the effects of the disease.

By “prevent” is meant to minimize the chance that a subject who has anincreased susceptibility for developing cancer will develop cancer.

By “specifically binds” is meant that an antibody recognizes andphysically interacts with its cognate antigen (for example, a hGC-1polypeptide) and does not significantly recognize and interact withother antigens; such an antibody may be a polyclonal antibody or amonoclonal antibody, which are generated by techniques that are wellknown in the art.

By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNAor RNA molecule of defined sequence that can base-pair to a second DNAor RNA molecule that contains a complementary sequence (the “target”).The stability of the resulting hybrid depends upon the extent of thebase-pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art. Probes or primers specificfor hGC-1 nucleic acids can (for example, genes and/or mRNAs) have atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% or 95% sequence complementarity, and should have at least 96%,97%, 98%, 99% or 100% sequence complementarity, to the region of thehGC-1 nucleic acid to which they hybridize. Probes, primers, andoligonucleotides may be detectably-labeled, either radioactively, ornon-radioactively, by methods well-known to those skilled in the art.Probes, primers, and oligonucleotides are used for methods involvingnucleic acid hybridization, such as: nucleic acid sequencing, reversetranscription and/or nucleic acid amplification by the polymerase chainreaction, single stranded conformational polymorphism (SSCP) analysis,restriction fragment polymorphism (RFLP) analysis, Southernhybridization, Northern hybridization, in situ hybridization,electrophoretic mobility shift assay (EMSA).

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a hGC-1 nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. See, for example, F. Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1998.

By “familial mutation” or “inherited mutation” is meant a mutation in anindividual that was inherited from a parent and that was present insomatic cells of the parent. By “sporadic mutation” or “spontaneousmutation” is meant a mutation in an individual that arose in theindividual and was not present in a parent of the individual.

The term “cancer,” when used herein refers to or describes thephysiological condition, such as in a mammalian subject, that istypically characterized by unregulated cell growth. Examples of cancerinclude but are not limited to myeloma, B-cell leukemia, and prostatecancer. While the term “cancer” as used herein is not limited to any onespecific form of the disease, it is believed that the methods of theinvention will be particularly effective for cancers which are found tobe accompanied by decreased levels of hGC-1 expression.

We have identified and characterized a hematopoietic granulocytecolony-stimulating factor (G-CSF)-induced olfactomedin-relatedglycoprotein and its corresponding gene, termed hGC-1.

The corresponding protein sequence of hGC-1 indicated that it is aglycoprotein of the olfactomedin family, which includes olfactomedin,TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes showcharacteristic tissue-restricted patterns of expression; the specifictissues that express these genes differ among the various familymembers. hGC-1 was strongly expressed in the prostate, small intestine,and colon, moderately expressed in the bone marrow and stomach, and notdetectable in other tissues. In vitro translation and ex vivo expressionshowed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mappedto chromosome 13q14.3.

The present data indicate that hGC-1 is primarily expressed as anextracellular olfactomedin-related glycoprotein during normalmyeloid-specific lineage differentiation, suggesting the possibility ofa matrix-related function for hGC-1 in differentiation.

The present invention provides an isolated nucleic acid encoding a humanhGC-1 gene comprising the nucleic acid set forth in the sequence listingSEQ ID NO:1 and described more fully herein infra. Further provided iscDNA clone comprising the sequence SEQ ID NO:2 and described more fullyherein infra.

Also provided is a mouse cDNA sequence comprising the sequence SEQ IDNO:3 and described more fully herein infra.

The present invention provides an isolated nucleic acid comprising a)the sequence of SEQ ID NO:1; b) the sequence of SEQ ID NO:2; c) thesequence of SEQ ID NO:3; d) a sequence complementary to any of a), b),or c); and e) a sequence of at least 10 contiguous nucleotides specificfor any of a)-d). A nucleic acid comprising one or more of the followingstructural characteristics: 2849 bp, 5 exons, a polyadenylation signalat bp 2818, an open reading frame of 1530 nucleotides, and having achromosomal locus of 13q14.3 is disclosed. A nucleic acid comprising anopen reading frame (ORF) of approximately 1530 nucleotides that encodesa protein of approximately 510 amino acids is provided. The nucleic acidcan be the genomic sequence of hGC-1 (e.g., SEQ ID NO:1) or it can bethe cDNA of hGC-1 (e.g., SEQ ID NO:2) or it can be the 1530 nucleotideORF of hGC-1 disclosed herein.

A nucleic acid, defined by a band at about 2.8 KB on a Northern blot,such as depicted in FIG. 4.

A nucleic acid with about 70% or greater homology to the nucleic acidcomprising a) the sequence of SEQ ID NO:1; b) the sequence of SEQ IDNO:2; c) the sequence of SEQ ID NO:3; d) a sequence complementary to anyof a), b), or c); or e) a sequence of at least 10 contiguous nucleotidesspecific for any of a)-d) is disclosed. A nucleic acid, which hybridizesto this nucleic acid under stringent conditions is additionallyprovided.

An isolated nucleic acid comprising a sequence of nucleotides encoding ahGC-1 protein is provided. The hGC-1 protein has the characteristics andstructure as described herein, and, for example, can have the an aminoacid sequence of SEQ ID NO:4. The nucleic acid can have the sequence ofSEQ ID NO:1 or SEQ ID NO:2 or the hGC-1 ORF shown in FIG. 2.

These nucleic acids can be cDNA sequences.

Compositions comprising these nucleic acids are disclosed.

An isolated gene is provided which comprises the nucleic acid of SEQ IDNO: 1, or an allelic variation thereof.

Compositions comprising the gene are disclosed.

Further, the present invention provides products encoded by the nucleicacids or gene of the present invention. These products can be peptides,polypeptides, or proteins. An example of a protein or polypeptide of thepresent invention is provided in sequence listing SEQ ID NO:4.

A purified protein consisting essentially of the sequence set forth inSEQ ID NO:4, or the sequence set forth in SEQ ID NO:4 having amino acidsubstitutions which conserve the biological or biochemical or structuralproperties of the amino acid sequence of SEQ ID NO:4, is disclosed. Theamino acid sequence set forth in SEQ ID NO:4 can be a human hGC-1protein.

A purified polypeptide having one or more of the followingcharacteristics: 510 amino acids, 6 N-linked glycosylation sites(motifs), extracellular protein, member of the olfactomedin family, andlacks a transmembrane domain is disclosed.

A polypeptide encoded by a nucleic acid of the invention can have adeletion in the region encoded by exon 5.

Compositions comprising the products encoded by the gene are disclosed.

A vector comprising the nucleic acid of the invention is disclosed. Thevectors are suitable for expressing the nucleic acid. A vector of thepresent invention expresses human hGC-1 protein in a host cell. Thehuman hGC-1 protein expressed may have the sequence of SEQ ID NO:4.

A cell comprising the vector is given. A cell transfected or transformedby the nucleic acid of the invention is disclosed.

A transgenic animal is disclosed. The transgenic animal comprises a cellthat has been transfected with the nucleic acid of the invention.

Further nucleic acids are described herein. These are nucleic acidscomprising the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12. These nucleic acids can be used asprimers. Compositions comprising these nucleic acids are also disclosed.

A purified antibody or fragment thereof, which specifically binds to thepolypeptide of the invention is provided. An antibody or fragmentthereof of the present invention has binding affinity for an antigenicregion of the polypeptide consisting essentially of SEQ ID NO:4 or otherpolypeptides of the invention.

Compositions comprising the antibody or fragment thereof are disclosed.

A method comprising identifying the expression of hGC-1 in a sample ofcells by detecting a nucleic acid specific for the nucleic acid of theinvention is provided. Expression of hGC-1 can indicate a cell is of thenormal myeloid lineage of cells.

A method is disclosed comprising identifying a patient at increased riskfor cancer by measuring the level of hGC-1 in the tissues of the patientthat normally expresses hGC-1, and comparing the measured hGC-1 levelwith hGC-1 levels found in healthy subjects, wherein a decrease in hGC-1in the patient indicates increased risk.

Also provided is a method of diagnosing cancer comprising detecting amutation of the hGC-1 gene in suspected cancer cells from a subject.

Further provided is a method of treating cancer comprising administeringto a subject a therapeutically effective amount of a non-mutant copy ofthe gene of the invention, fragment of the gene, nucleic acid of theinvention, or polypeptide of the invention to affected cells.

Additionally, a method of preventing cancer in a subject comprisingadministering a preventative amount of the hGC-1 gene, fragment of thegene, or nucleic acid of the invention to cells of the subject having amutant copy of the gene is provided.

Still further provided is a method of preventing cancer in a subjectcomprising administering a preventative amount of a polypeptide of theinvention to a subject.

A method of detecting antibodies that bind to hGC-1 in a biologicalsample is disclosed, comprising the steps of: a) contacting thepolypeptide of the invention with the biological sample suspected ofcontaining the polypeptide antibodies under conditions that allow forformation of an antibody-antigen complex; and b) detecting theantibody-antigen complex, whereby the presence of the complex indicatesthe presence of antibodies that bind to hGC-1.

A method of detecting hGC-1 or an antigenic fragment thereof in a sampleis also disclosed, comprising a) contacting the sample with an antibodywhich selectively binds with the polypeptide of the invention and b)detecting binding of the antibody and antigen, whereby the presence ofthe complex indicates the presence of antibodies that bind to hGC-1.

The invention discloses a method for detecting the presence of hGC-1antibodies comprising a) binding an hGC-1 polypeptide to a substrate, b)contacting the bound polypeptide with a sample, c) adding secondaryantibodies which bind with the hGC-1 antibodies and which are labeled orbound with a detectable moeity, and d) visualizing the secondaryantibody as well.

A method of detecting a mutant hGC-1 gene comprising a) contacting thesample with an antibody which selectively binds with a mutant hGC-1 andb) detecting binding of the antibody and antigen, whereby the presenceof the complex indicates the presence of antibodies that bind to amutant hGC-1 is provided.

The invention also provides kits comprising the compositions of thepresent invention. Such kits include the following. A kit comprising apackaging, containing the nucleic acid, the polypeptide, or antibodiesof the present invention. A kit for amplifying hGC-1 comprising apackaging, containing, separately packaged a forward primer and areverse primer comprising nucleic acids of the invention are alsodisclosed. A diagnostic kit for diagnosing cancer or identifying asubject with an increased susceptibility for cancer comprising apackaging, containing, separately packaged reagents for detecting amutant hGC-1 polypeptide or mutated hGC-1 nucleic acid in the subjectare further disclosed.

mRNA differential display analysis was used to identify lineage-specificexpressed genes in induced early precursors of blood cell lineages. Oneclone specifically induced by G-CSF, hGC-1, was further characterized.The corresponding amino acid sequence of hGC-1 indicated that the hGC-1protein is a glycoprotein of the olfactomedin family. The hGC-1 geneshowed tissue-restricted patterns of expression characteristic of theolfactomedin family. hGC-1 was strongly expressed in the prostate, smallintestine, and colon, moderately expressed in the bone marrow andstomach, and not detectable in other tissues. In vitro translation andex vivo expression showed hGC-1 to be an N-linked glycoprotein. ThehGC-1 gene locus mapped to chromosome 13q14.3.

The following compositions and methods are provided.

Compositions

The compositions of the present invention include, but are not limitedto, the following.

Suitable experimental methods for making the compositions of theinvention could be determined by one of ordinary skill in the art.Methods for making specific and preferred compositions of the presentinvention are described in detail in the Examples below.

Although any methods and materials capable of producing the compositionsof the present invention can be used, such as those similar orequivalent to those described herein, in the preparation, practice ortesting of the present invention, those methods and materials used todate by the inventors are described herein.

Gene/Nucleic Acids/cDNA

The gene, hGC-1 is provided. An example of an hGC-1 gene is the nucleicacid of SEQ ID NO:1. Allelic variations of hGC-1 are included in thisinvention. Compositions comprising the gene, or its allelic variants,are also included.

The invention provides isolated nucleic acids that comprise, consistessentially of or consist of the nucleic acids set forth in the SequenceListing as SEQ ID NO:1 (corresponding to a genomic DNA for an hGC-1gene, also depicted in FIG. 3A), SEQ ID NO:2 (corresponding to a cDNAencoding a human hGC-1) and SEQ ID NO:3 (corresponding to a nucleic acidencoding a mouse GC-1 cDNA).

The nucleic acids, gene or its allelic variants may be synthesized,isolated, purified, or otherwise made according to methods generallyknown by one of skill in the art by standard methods. For example, seeSambrook et al., “Molecular Cloning: A Laboratory Manual” 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and Kunkel etal. Methods Enzymol. 1987:154:367 (1987). Compositions comprising thegene, or its variants, may also be made according to methods generallyknown by one of skill in the art, for example, by simply adding thegene, or its variants, to another composition, such as a carrier. Thenucleic acids, gene, or its variants, may likewise be added to otherknown or yet to be discovered compositions.

Additionally, contemplated by the present invention are nucleic acids,from any desired species, such as mammalian and more specifically human,having 99.9%, 99.7%, 99.5%, 99.3%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% overall homology orhomology in the region being compared to the same region of a nucleicacid set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or to a nucleicacid encoding the polypeptide set forth in SEQ ID NO:4, or to allelicvariants or homologs thereof. The assessment of homology is preferablybased on a base-by-base comparison of the regions being compared. Thesegenes and nucleic acids can be synthesized or obtained by the samemethods used to isolate homologs, with stringency of hybridization andwashing, if desired, reduced accordingly as homology desired isdecreased, and further, depending upon the G-C or A-T richness of anyarea wherein variability is searched for. Allelic variants of any of thepresent genes and nucleic acids or of their homologs can readily beisolated and sequenced by screening additional libraries following theexamples given herein and procedures well known in the art.

The gene or nucleic acid encoding any selected protein of the presentinvention can be any nucleic acid that functionally encodes thatprotein. A nucleic acid encoding a selected protein can readily bedetermined based upon the amino acid sequence of the selected protein,and, clearly, many nucleic acids will encode any selected protein.

The present invention additionally provides a nucleic acid thatselectively hybridizes under stringent conditions with a nucleic acidset forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Thishybridization can be specific. The degree of complementarity between thehybridizing nucleic acid and the sequence to which it hybridizes shouldbe at least enough to exclude hybridization with a nucleic acid encodingan unrelated protein. Thus, a nucleic acid that selectively hybridizeswith a nucleic acid of a present protein coding sequence will notselectively hybridize under stringent conditions with a nucleic acid fora different, unrelated protein, and vice versa. The temperature and saltconditions are readily determined empirically in preliminary experimentsin which samples of reference DNA immobilized on filters are hybridizedto a labeled nucleic acid of interest and then washed under conditionsof different stringencies.

Hybridization temperatures are typically higher for DNA-RNA and RNA-RNAhybridizations. The washing temperatures can be used as described aboveto achieve selective stringency, as is known in the art. (See, forexample, Sambrook et al., “Molecular Cloning: A Laboratory Manual” 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) andKunkel et al. Methods Enzymol. 1987:154:367 (1987)). Nucleic acidfragments that selectively hybridize to any given nucleic acid can beused, e.g., as primers and or probes for further hybridization or foramplification methods (e.g., polymerase chain reaction (PCR), ligasechain reaction (LCR)).

cDNA of the present invention comprises SEQ ID NO:2 or SEQ ID NO:3.Compositions comprising the cDNA are also included.

The cDNA may be synthesized, isolated, purified, or otherwise obtainedaccording to methods generally known by one of skill in the art.Compositions comprising the cDNA may also be made according to methodsgenerally known by one of skill in the art, for example, by simplyadding the cDNA to another composition, such as a carrier. The cDNA maylikewise be added to other known or yet to be discovered compositions.

One skilled in the art will appreciate that the cDNA or cDNA fragments(probes, primers etc.) encoding hGC-1 provide information with which thegenomic nucleic acids corresponding to this cDNA, or genomic variants ofthis gene can be isolated. For example, primers for amplifying the hGC-1gene that encodes the hGC-1 protein can be designed using the sequenceinformation disclosed in SEQ ID NO:1 or 2. Alternatively, those samedisclosed nucleic acids can be used to design probes for detecting anucleic acid containing all or part of the genomic nucleic acid in agenomic library.

The invention includes fragments of the nucleic acids. The fragmentsmay, for example, be probes or primers of expression sequences. Forexample, the fragments may comprise SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

Examples of primers according to the present invention comprise SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ IDNO:12.

Compositions comprising the primers are also included.

The primers may be synthesized or otherwise made according to methodsgenerally known by one of skill in the art. Compositions comprising oneof or both of the primers may also be made according to methodsgenerally known by one of skill in the art, for example, by simplyadding the primer(s) to another composition, such as a carrier. Theprimer(s) may likewise be added to other known or yet to be discoveredcompositions.

The present invention also contemplates polynucleotide probes fordetecting a hGC-1 gene, wherein the polynucleotide probe hybridizes tothe nucleotide sequence set forth in the Sequence Listing as SEQ IDNO:1, SEQ ID NO:2.

As used herein, the term “polynucleotide probe” refers to a nucleic acidfragment that selectively hybridizes under stringent conditions with anucleic acid comprising a nucleic acid set forth in a sequence listedherein. This hybridization must be specific. The degree ofcomplementarity between the hybridizing nucleic acid and the sequence towhich it hybridizes should be at least enough to exclude hybridizationwith a nucleic acid encoding an unrelated protein.

Thus, allelic variants can be identified and isolated by nucleic acidhybridization techniques. Probes selective to the nucleic acid set forthin the Sequence Listing as SEQ ID NO:1 or SEQ ID NO:2 can be synthesizedand used to probe the nucleic acid from various cells, tissues,libraries etc. High sequence complementarity and stringent hybridizationconditions can be selected such that the probe selectively hybridizes toallelic variants of the sequence set forth in the Sequence Listing asSEQ ID NO:1 or SEQ ID NO:2. For example, the selectively hybridizingnucleic acids of the invention can have at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% and 99% complementarity with the segment of thesequence to which it hybridizes. The nucleic acids can be at least 10,12, 50, 100, 150, 200, 300, 500, 750, 1000, 1250, 1500, 1750, 2000,2250, 2500, 2750, 2800, 2849, 2861, or 2870 nucleotides in length. Thus,the nucleic acid can be a coding sequence for a hGC-1 protein orfragments thereof that can be used as a probe or primer for detectingthe presence of these genes. If used as primers, the invention providescompositions including at least two nucleic acids which hybridize withdifferent regions so as to amplify a desired region. Several primers areprovided below as examples of amplification primers. Depending on thelength of the probe or primer, target region can range between 90%complementary bases and full complementarity and still hybridize understringent conditions. For example, for the purpose of detecting thepresence of an allelic variant of the sequence set forth SEQ ID NO:1 orSEQ ID NO:2, the degree of complementarity between the hybridizingnucleic acid (probe or primer) and the sequence to which it hybridizesis at least enough to distinguish hybridization with a nucleic acid fromother species. The invention provides examples of nucleic acids uniqueto SEQ ID NO:1 or SEQ ID NO:2 so that the degree of complementarityrequired to distinguish selectively hybridizing from nonselectivelyhybridizing nucleic acids under stringent conditions can be clearlydetermined for each nucleic acid.

“Stringent conditions” refers to the washing conditions used in ahybridization protocol. In general, the washing conditions should be acombination of temperature and salt concentration chosen so that thedenaturation temperature is approximately 5-20° C. below the calculatedT_(m) of the nucleic acid hybrid under study. The temperature and saltconditions are readily determined empirically in preliminary experimentsin which samples of reference DNA immobilized on filters are hybridizedto the probe or protein coding nucleic acid of interest and then washedunder conditions of different stringencies. The T_(m) of such anoligonucleotide can be estimated by allowing 2° C. for each A or Tnucleotide, and 4° C. for each G or C. For example, an 18 nucleotideprobe of 50% G+C would, therefore, have an approximate T_(m) of 54° C.

The present invention also contemplates any unique fragment of the geneor of the nucleic acids set forth in SEQ ID NO:1 or SEQ ID NO:2. To beunique, the fragment must be of sufficient size to distinguish it fromother known sequences, most readily determined by comparing any nucleicacid fragment to the nucleotide sequences of nucleic acids in computerdatabases, such as GenBank. Such comparative searches are standard inthe art. Typically, a unique fragment, useful as a primer or probe, willbe at least about 20 to about 25 nucleotides in length, depending uponthe specific nucleotide content of the sequence. Additionally, fragmentscan be, for example, at least about 10, 12, 30, 40, 50, 75, 100, 200,210, 211, 212, 213, 214, 215, 216, 220, 230, 240, 245, 246, 247, 248,249, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500,1750, 2000, 2250, 2500, 2750, 2849, 2861 nucleotides in length or anynumber in between. All of the genes, nucleic acids, and fragments of thegenes' and nucleic acids disclosed and contemplated herein can be singleor double stranded, depending upon the purpose for which it is intended.

The invention also contemplates compounds comprising the genes, nucleicacids, and fragments of the hGC-1 genes and nucleic acids as disclosedand contemplated herein. For example, a compound comprising a nucleicacid can be a derivative of a typical nucleic acid such as nucleic acidswhich are modified to contain a terminal or internal reporter moleculeand/or those nucleic acids containing non-typical bases or sugars. Thesereporter molecules include, but are not limited to, isotopic andnon-isotopic reporters. Examples include, a FLAG tag or a human IgG Fc.Therefore, any molecule which may aid in detection, amplification,replication, expression, purification, uptake, etc. may be added to thenucleic acid construct.

The genes and nucleic acids provided for by the present invention may beobtained in any number of ways. For example, a DNA molecule encoding ahGC-1 protein can be isolated from the organism in which it is normallyfound. For example, a genomic DNA or cDNA library can be constructed andscreened for the presence of the gene or nucleic acid of interest.Methods of constructing and screening such libraries are well known inthe art and kits for performing the construction and screening steps arecommercially available (for example, Stratagene Cloning Systems, LaJolla, Calif.). Once isolated, the gene or nucleic acid can be directlycloned into an appropriate vector, or if necessary, be modified tofacilitate the subsequent cloning steps. Such modification steps areroutine, an example of which is the addition of oligonucleotide linkerswhich contain restriction sites to the termini of the nucleic acid.General methods are set forth in Sambrook et al., “Molecular Cloning, aLaboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

Once the gene or nucleic acid sequence of the desired hGC-1 protein isobtained, the sequence encoding specific amino acids can be modified orchanged at any particular amino acid position by techniques well knownin the art. For example, PCR primers can be designed which span theamino acid position or positions and which can substitute any amino acidfor another amino acid. Then a nucleic acid can be amplified andinserted into the wild-type hGC-1 protein coding sequence in order toobtain any of a number of possible combinations of amino acids at anyposition of the hGC-1 protein. Alternatively, one skilled in the art canintroduce specific mutations at any point in a particular nucleic acidsequence through techniques for point mutagenesis. General methods areset forth in Smith, M. “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462(1985) and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605-610 (1991). Techniquessuch as these can also be used to modify the genes or nucleic acids inregions other than the coding regions, such as the promoter regions forthe hGC-1 protein. Likewise, these techniques can be used to alter thecoding sequence without altering the amino acid sequence that isencoded.

Another example of a method of obtaining a DNA molecule encoding aspecific hGC-1 protein, polypeptide, or peptide is to synthesize arecombinant DNA molecule which encodes the hGC-1 protein. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein region are readilyobtainable through automated DNA synthesis. A nucleic acid for onestrand of a double-stranded molecule can be synthesized and hybridizedto its complementary strand. One can design these oligonucleotides suchthat the resulting double-stranded molecule has either internalrestriction sites or appropriate 5′ or 3′ overhangs at the termini forcloning into an appropriate vector. Double-stranded molecules coding forrelatively large proteins can readily be synthesized by firstconstructing several different double-stranded molecules that code forparticular regions of the protein, followed by ligating these DNAmolecules together. For example, Cunningham, et al., “Receptor andAntibody Epitopes in Human Growth Hormone Identified by Homolog-ScanningMutagenesis,” Science, 243:1330-1336 (1989), have constructed asynthetic gene encoding the human growth hormone gene by firstconstructing overlapping and complementary synthetic oligonucleotidesand ligating these fragments together. See also, Ferretti, et al., Proc.Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pairsynthetic bovine rhodopsin gene from synthetic oligonucleotides isdisclosed. By constructing a hGC-1 protein in this manner, one skilledin the art can readily obtain any particular hGC-1 protein with desiredamino acids at any particular position or positions within the hGC-1protein. See also, U.S. Pat. No. 5,503,995, which describes an enzymetemplate reaction method of making synthetic genes. Techniques such asthis are routine in the art and are well-documented. These nucleic acidsor fragments of a nucleic acid encoding a hGC-1 protein can then beexpressed in vivo or in vitro as discussed below.

Amino Acid Sequence/Polypeptide/Protein

The invention provides the hGC-1 protein. The amino acid sequence ofhGC-1 can correspond to the nucleotide sequence of hGC-1 as describedherein. The protein of the present invention comprises the amino acidsequence of SEQ ID NO:4. The invention includes the immature and matureprotein or polypeptides. Compositions comprising the protein, amino acidsequence, or polypeptides are also included.

The amino acid sequence, protein, and peptides may be synthesized,isolated, purified, or otherwise obtained according to methods generallyknown by one of skill in the art. Compositions comprising the amino acidsequence, protein, and peptides may also be made according to methodsgenerally known by one of skill in the art, for example, by simplyadding the amino acid sequence, protein, or polypeptide to anothercomposition, such as a carrier. The amino acid sequence, protein, andpolypeptide may likewise be added to other known or yet to be discoveredcompositions, such as other proteins or polypeptides.

FIG. 6 and its corresponding description indicate the predictedsecondary structure of the hGC-1 protein and its homology toolfactomedin-related proteins. It is understood that a polypeptidehaving one or more of the defined secondary structural characteristicsis within the scope of the invention. The polypeptide should alsopossess at least one of the functions of hGC1.

The invention also provides polypeptides encoded by the nucleic acid setforth in SEQ ID NO:1 or SEQ ID NO:2, and the polypeptides set forth theSequence Listing as SEQ ID NO:4. The invention also provides fragmentsof unmodified and modified hGC-1 proteins. The fragments can be specificfor hGC-1 or specific for the hGC-1 shown in SEQ ID NO:4. The fragmentscan be at least 5 amino acids long, and any greater length up to oneamino acid less than the full-length protein. The polypeptide fragmentsof the present invention can be recombinant proteins obtained by cloningnucleic acids encoding the polypeptide in an expression system capableof producing the polypeptide fragments thereof. For example, one skilledin the art can determine the active regions of a hGC-1 protein which caninteract with another protein and cause a biological effect associatedwith the hGC-1 protein. In one example, amino acids found to notcontribute to either the activity, binding specificity, or otherbiological effect associated with the hGC-1 protein can be deletedand/or substituted without a loss in the respective activity. Thefragments, whether attached to other sequences or not, can also includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acid residues, provided theactivity of the peptide is not significantly altered or impaired.

The fragments of hGC-1 protein disclosed herein are identified in FIG. 6by selecting fragments (regions) that are not 100% identical to theregion of a related protein. The fragments can exclude the stretch of223 amino acids in the carboxy terminus of hGC-1 which are highlyconserved among other olfactomedin proteins, but, to the extent thatthere is any region lacking 100% identity, that region can constitute afragment of the invention.

Further contemplated are polypeptides encoded by fragments of the hGC-1nucleic acids provided herein. It is noted that the hGC-1 proteinfragment and that other examples of hGC-1 and hGC-1 fragments havingslightly different sequences may be found in nature using routineprotocols or generated by design. Additional fragments of hGC-1 can beidentified based on correspondence to functional regions of otherpreviously known members of the olfactomedin family.

By “active fragment” is meant a subpart of a whole hGC-1 protein thatexhibits an activity of hGC-1.

The hGC-1 polypeptides of this invention can also be fused to anotherprotein such as alkaline phosphatase for detection methods. This fusionpolypeptide can be utilized in an ELISA or Western blot to detect thereceptor for hGC-1. The hGC-1-alkaline phosphatase (hGC-1AP)polypeptides of this invention can be used to measure hGC-1 in anELISA-based assay. For example, plates can be coated with an anti-hGC-1antibody. Samples can then be added to the plates that contain a givenamount of hGC-1AP which will bind to the plates. Therefore, in theabsence of antigen, hGC-1AP should bind to the plates and produce themaximal amount of alkaline phosphatase activity. Upon addition ofsamples containing hGC-1, the antigen may compete with hGC-1AP for thehGC-1 antibody on the plates. The presence of antigen will competitivelyinhibit the binding of hGC-1AP.

Once the gene or nucleic acid sequence of the desired hGC-1 protein isobtained, the sequence encoding specific amino acids can be modified orchanged at any particular amino acid position by techniques well knownin the art. For example, PCR primers can be designed which span theamino acid position or positions and which can substitute any amino acidfor another amino acid. Then a nucleic acid can be amplified andinserted into the wild-type hGC-1 protein coding sequence in order toobtain any of a number of possible combinations of amino acids at anyposition of the hGC-1 protein. Alternatively, one skilled in the art canintroduce specific mutations at any point in a particular nucleic acidsequence through techniques for point mutagenesis. General methods areset forth in Smith, M. “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462(1985) and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605-610 (1991). Techniquessuch as these can also be used to modify the genes or nucleic acids inregions other than the coding regions, such as the promoter regions forthe hGC-1 protein. Likewise, these techniques can be used to alter thecoding sequence without altering the amino acid sequence that isencoded.

Another example of a method of obtaining a DNA molecule encoding aspecific hGC-1 protein is to synthesize a recombinant DNA molecule whichencodes the hGC-1 protein. For example, oligonucleotide synthesisprocedures are routine in the art and oligonucleotides coding for aparticular protein region are readily obtainable through automated DNAsynthesis. A nucleic acid for one strand of a double-stranded moleculecan be synthesized and hybridized to its complementary strand. One candesign these oligonucleotides such that the resulting double-strandedmolecule has either internal restriction sites or appropriate 5′ or 3′overhangs at the termini for cloning into an appropriate vector.Double-stranded molecules coding for relatively large proteins canreadily be synthesized by first constructing several differentdouble-stranded molecules that code for particular regions of theprotein, followed by ligating these DNA molecules together. For example,Cunningham, et al., “Receptor and Antibody Epitopes in Human GrowthHormone Identified by Homolog-Scanning Mutagenesis,” Science,243:1330-1336 (1989), have constructed a synthetic gene encoding thehuman growth hormone gene by first constructing overlapping andcomplementary synthetic oligonucleotides and ligating these fragmentstogether. See also, Ferretti, et al., Proc. Nat. Acad. Sci. 82:599-603(1986), wherein synthesis of a 1057 base pair synthetic bovine rhodopsingene from synthetic oligonucleotides is disclosed. By constructing ahGC-1 protein in this manner, one skilled in the art can readily obtainany particular hGC-1 protein with desired amino acids at any particularposition or positions within the hGC-1 protein. See also, U.S. Pat. No.5,503,995, which describes an enzyme template reaction method of makingsynthetic genes. Techniques such as this are routine in the art and arewell-documented. These nucleic acids or fragments of a nucleic acidencoding a hGC-1 protein can then be expressed in vivo or in vitro asdiscussed below.

The hGC-1 polypeptides can be obtained in any of a number of procedureswell known in the art. One method of producing a polypeptide is to linktwo peptides or polypeptides together by protein chemistry techniques.For example, peptides or polypeptides can be chemically synthesizedusing currently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to a particular protein can be synthesized by standardchemical reactions. For example, a peptide or polypeptide can besynthesized and not cleaved from its synthesis resin whereas the otherfragment of a hybrid peptide can be synthesized and subsequently cleavedfrom the resin, thereby exposing a terminal group which is functionallyblocked on the other fragment. By peptide condensation reactions, thesetwo fragments can be covalently joined via a peptide bond at theircarboxyl and amino termini, respectively, to form a larger polypeptide.(Grant, “Synthetic Peptides: A User Guide,” W. H. Freeman and Co., N.Y.(1992) and Bodansky and Trost, Ed., “Principles of Peptide Synthesis,”Springer-Verlag Inc., N.Y. (1993)). Alternatively, the peptide orpolypeptide can be independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides may be linkedto form a larger protein via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentscan allow relatively short peptide fragments to be joined to producelarger peptide fragments, polypeptides or whole protein domains(Abrahmsen et al. Biochemistry, 30:4151 (1991)). Alternatively, nativechemical ligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.“Synthesis of Proteins by Native Chemical Ligation,” Science,266:776-779 (1994)). The first step is the chemoselective reaction of anunprotected synthetic peptide-α-thioester with another unprotectedpeptide segment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis et al. FEBSLett., 307:97 (1987), Clark-Lewis et al., J. Biol. Chem., 269:16075(1994), Clark-Lewis et al. Biochemistry, 30:3128 (1991), and Rajarathnamet al. Biochemistry, 29:1689 (1994)).

Alternatively, unprotected peptide segments can be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (non-peptide) bond (Schnolzer et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton et al.“Techniques in Protein Chemistry IV,” Academic Press, New York, pp.257-267 (1992)).

Vector/Cells/Cell Line/Transgenic Animal

The invention provides a vector comprising the nucleic acids of theinvention. Examples of the vectors of the invention include thosecomprising the nucleotide sequence of SEQ ID NO:1. SEQ ID NO:2, or SEQID NO:3. The vector can further comprise any regulatory elementsnecessary for expression of the sequence in a cell. Compositionscomprising the vector are also included.

The vector may be synthesized or otherwise made according to methodsgenerally known by one of skill in the art Compositions comprising thevector may also be made according to methods generally known by one ofskill in the art, for example, by simply adding the vector to anothercomposition, such as a carrier. The vector may likewise be added toother known or yet to be discovered compositions.

The invention also provides for the isolated nucleic acids of SEQ IDNO:1 and SEQ ID NO:2 in a vector suitable for expressing the nucleicacid. Once a nucleic acid encoding a particular hGC-1 protein ofinterest, or a region of that nucleic acid, is constructed, modified, orisolated, that nucleic acid can then be cloned into an appropriatevector, which can direct the in vivo or in vitro synthesis of thatwild-type and/or modified hGC-1 protein. The vector is contemplated tohave the necessary functional elements that direct and regulatetranscription of the inserted gene, or nucleic acid. These functionalelements include, but are not limited to, a promoter, regions upstreamor downstream of the promoter, such as enhancers that may regulate thetranscriptional activity of the promoter, an origin of replication,appropriate restriction sites to facilitate cloning of inserts adjacentto the promoter, antibiotic resistance genes or other markers which canserve to select for cells containing the vector or the vector containingthe insert, RNA splice junctions, a transcription termination region, orany other region which may serve to facilitate the expression of theinserted gene or hybrid gene. (See generally, Sambrook et al.).

The invention includes cells transformed or transfected by a vector ofthe present invention. The cells may be in vitro or in vivo. The cellsmay comprise a transgenic animal, such as a mouse. The inventionincludes a knock-out mouse which can be used to test compositions of thepresent invention or other compositions related to expression of hGC-1.

The cells may be transformed or transfected by any method known in theart that is capable of transforming or transfecting the chosen cells.

This invention also contemplates producing a selected cell line or anon-human transgenic animal model for the analysis of the function of agene comprising introducing into an embryonic stem cell a vector havinga selectable marker which, when the vector is inserted within a gene,the inserted vector can inhibit the expression of the gene, selectingembryonic stem cells expressing the selectable marker, excising thevector from the embryonic stem cells expressing the selectable markersuch that host DNA from the gene is linked to the excised vector,sequencing the host DNA in the excised vector, comparing the sequence ofthe host DNA to known gene sequences to determine which host DNA is froma gene for which a model for the analysis of the function the gene isdesired, selecting the embryonic stem cell containing the inhibited genefor which a model for the analysis of gene function is desired, andforming a cell line or a non-human transgenic animal from the selectedembryonic stem cell.

Transgenic animals are provided, which either overproduce thepolypeptides of this invention or fail to produce the polypeptides ofthis invention in a functional form. For example, a transgenic animalwhich overproduces a hGC-1 of this invention can be produced accordingto methods well known in the art, whereby a nucleic acid encoding hGC-1is introduced into embryonic stem cells, at which stage it isincorporated into the germline of the animal, resulting in theproduction of hGC-1 in the transgenic animal in increased amountsrelative to a normal animal of the same species. One skilled in the artcan determine if overproduction or underproduction of hGC-1 results inaltered characteristics, such as predisposition to prostate cancer.Specifically, it has been shown that the hGC-1 gene is normallyexpressed in prostate epithelial cells, but not in prostate cancertissues, nor in samples derived from men with benign prostatehypertrophy (BPH). Furthermore, the gene was not expressed in 4 prostatecancer cell lines (Example 12). The mutation(s) in hGC1 associated withcancer can be used to make a transgenic mouse model of the relevantcancer, for example, to search for anti-cancer drugs. Such a transgenicanimal can be used to clarify the process of carcinogenesis from normalto pre-cancerous to cancer, thereby allowing targets for pharmaceuticalintervention.

A transgenic animal in which the expression of hGC-1, for example, istissue specific is also contemplated for this invention. For example,transgenic animals that express or overexpress these genes at specificsites, such as bone marrow, gastrointestinal tract, or genitourinarytract can be produced by introducing a nucleic acid into the embryonicstem cells of the animal, wherein the nucleic acid is under the controlof a specific promoter which allows expression of the nucleic acid inspecific types of cells (e.g., a bone marrow cell, gastrointestinaltract cell or genitourinary tract cell promoter which allows expressiononly in those cells). One skilled in the art can determine if atissue-specific alteration in hGC-1 expression results in altered cancerincidence.

Alternatively, the transgenic animal of this invention can be a “knockout” animal (see, e.g., Willnow et al., 1996), which can be an animalthat, for example, normally produces hGC-1 but has been altered toprevent the expression of the animal's nucleic acid which encodes hGC-1,thereby resulting in an animal which does not produce hGC-1 in afunctional form. Such an animal may lack the ability to express all ofthe nucleic acids encoding hGC-1 or the transgenic animal may lack theability to express some (one or more than one) but not all of thenucleic acids encoding the hGC-1.

For example, the transgenic “knock out” animal of this invention canhave the expression of a gene or genes knocked out in specific tissues.This approach obviates viability problems that can be encountered if theexpression of a widely expressed gene is completely abolished in alltissues.

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxy-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, et al.,“α-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae.” Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression of activeproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation may be used forother cellular hosts.

Alternative vectors for the expression of genes or nucleic acids inmammalian cells, those similar to those developed for the expression ofhuman gamma-interferon, tissue plasminogen activator, clotting FactorVIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophilmajor basic protein, can be employed. Further, the vector can includeCMV promoter sequences and a polyadenylation signal available forexpression of inserted nucleic acids in mammalian cells (such as COS-7).

Insect cells also permit the expression of mammalian proteins.Recombinant proteins produced in insect cells with baculovirus vectorsundergo post-translational modifications similar to that of wild-typeproteins. Briefly, baculovirus vectors useful for the expression ofactive proteins in insect cells are characterized by insertion of theprotein coding sequence downstream of the Autographica californicanuclear polyhedrosis virus (AcNPV) promoter for the gene encodingpolyhedrin, the major occlusion protein. Cultured insect cells such asSpodoptera frugiperda cell lines are transfected with a mixture of viraland plasmid DNAs and the viral progeny are plated. Deletion orinsertional inactivation of the polyhedrin gene results in theproduction of occlusion negative viruses which form plaques that aredistinctively different from those of wild-type occlusion positiveviruses. These distinctive plaque morphologies allow visual screeningfor recombinant viruses in which the AcNPV gene has been replaced with ahybrid gene of choice. High quantity expression and production of ahGC-1 protein can also be achieved by transgenic animal technology bywhich animals can be made to produce hGC-1 in serum, milk, etc. in largeamounts.

The invention also provides for the vectors containing the contemplatednucleic acids in a host suitable for expressing the nucleic acids. Thevectors containing the nucleic acid segments of interest can betransferred into host cells by well-known methods, which vary dependingon the type of cellular host. For example, calcium chloridetransformation, transduction, and electroporation are commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofection mediated transfection or electroporation may be used forother cellular hosts.

Alternatively, the genes or nucleic acids of the present invention canbe operatively linked to one or more of the functional elements thatdirect and regulate transcription of the inserted gene as discussedabove and the gene or nucleic acid can be expressed. For example, a geneor nucleic acid can be operatively linked to a bacterial or phagepromoter and used to direct the transcription of the gene or nucleicacid in vitro. A further example includes using a gene or nucleic acidprovided herein in a coupled transcription-translation system where thegene directs transcription and the RNA thereby produced is used as atemplate for translation to produce a polypeptide. One skilled in theart will appreciate that the products of these reactions can be used inmany applications such as using labeled RNAs as probes and usingpolypeptides to generate antibodies or in a procedure where thepolypeptides are being administered to a cell or a subject.

Expression of the gene or nucleic acid, in combination with a vector,can be by either in vivo or in vitro. In vivo synthesis comprisestransforming prokaryotic or eukaryotic cells that can serve as hostcells for the vector. Alternatively, expression of the gene or nucleicacid can occur in an in vitro expression system. For example, in vitrotranscription systems are commercially available which are routinelyused to synthesize relatively large amounts of mRNA. In such in vitrotranscription systems, the nucleic acid encoding the hGC-1 protein wouldbe cloned into an expression vector adjacent to a transcriptionpromoter. For example, the Bluescript II cloning and expression vectorscontain multiple cloning sites which are flanked by strong prokaryotictranscription promoters. (Stratagene Cloning Systems, La Jolla, Calif.).Kits are available which contain all the necessary reagents for in vitrosynthesis of an RNA from a DNA template such as the Bluescript vectors.(Stratagene Cloning Systems, La Jolla, Calif.). RNA produced in vitro bya system such as this can then be translated in vitro to produce thedesired hGC-1 protein. (Stratagene Cloning Systems, La Jolla, Calif.).

Antibodies/Ligands

The invention includes isolated antibodies or fragments thereof withbinding affinity for antigenic region(s) of a polypeptide or nucleicacid of the present invention. Compositions comprising the antibodiesare also included.

The antibodies may be isolated, purified, synthesized or otherwiseobtained according to methods generally known by one of skill in theart. Compositions comprising the antibodies may also be made accordingto methods generally known by one of skill in the art, for example, bysimply adding the antibodies to another composition, such as a carrier.The antibodies may likewise be added to other known or yet to bediscovered compositions, such as other antibodies.

Also provided herein are purified antibodies that selectively orspecifically bind to the hGC-1 polypeptides provided and contemplatedherein, for example, purified antibodies which selectively orspecifically bind to a polypeptide encoded by the nucleic acid set forthin any of SEQ ID NO:1 or SEQ ID NO:2, and purified antibodies whichselectively or specifically bind to the polypeptide set forth in SEQ IDNO:4. The antibody (either polyclonal or monoclonal) can be raised toany of the polypeptides provided and contemplated herein, bothnaturally-occurring and recombinant polypeptides, and immunogenicfragments, thereof. The antibody can be used in techniques or proceduressuch as diagnostics, treatment, or vaccination. Anti-idiotypicantibodies and affinity-matured antibodies are also considered.

Antibodies against hGC-1 are provided. Antibodies can be raised againstthe whole hGC-1 molecule or against immunogenic fragments of it.

The purified antibodies of this invention include monoclonal antibodieswhich can be used for diagnostic or analytical purposes. For example,the monoclonal antibody could be utilized in a clinical testing kit tomonitor levels of hGC-1 in human tissues or secretions.

The invention includes ligands that specifically bind to a polypeptideof the present invention. Compositions comprising the ligands are alsoincluded.

The ligands may be synthesized or otherwise made according to methodsgenerally known by one of skill in the art. Compositions comprising theligands may also be made according to methods generally known by one ofskill in the art, for example, by simply adding the ligands to anothercomposition, such as a carrier. The ligands may likewise be added toother known or yet to be discovered compositions.

Antibodies can be made by many well-known methods (See, e.g. Harlow andLane, “Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can beinjected into an animal in an amount and in intervals sufficient toelicit an immune response. Antibodies can either be purified directly,or spleen cells can be obtained from the animal. The cells can thenfused with an immortal cell line and screened for antibody secretion.The antibodies can be used to screen nucleic acid clone libraries forcells secreting the antigen. Those positive clones can then besequenced. (See, for example, Kelly et al. Bio/Technology, 10:163-167(1992); Bebbington et al. Bio/Technology, 10:169-175 (1992)). Humanizedand chimeric antibodies are also contemplated in this invention.Heterologous antibodies can be made by well-known methods (See, forexample, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016; 5,770,429; 5,789,650; and 5,814,318).

The specifically binding polypeptide or antibody can interact in abinding reaction, which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies boundto a particular protein do not bind in a significant amount to otherproteins present in the sample. Selective binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select antibodies that selectively bind with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies selectively immunoreactive with a protein. SeeHarlow and Lane “Antibodies, A Laboratory Manual” Cold Spring HarborPublications, New York, (1988), for a description of immunoassay formatsand conditions that could be used to determine selective binding.

Kits

The invention includes kits comprising the compositions of the presentinvention. The compositions may be separately packaged and provided inkits for use in various methods, such as the diagnostic method describedbelow.

The present invention provides a kit for detecting the binding of anantibody to the hGC-1 or the hGC-1 receptor, or a fragment thereof.Particularly, the kit can detect the presence of an antigen specificallyreactive with the antibody or an immunoreactive fragment thereof. Thekit can include an antibody bound to a substrate, a secondary antibodyreactive with the antigen and a reagent for detecting a reaction of thesecondary antibody with the antigen. Such a kit can be an ELISA kit andcan comprise the substrate, primary and secondary antibodies whenappropriate, and any other necessary reagents such as detectablemoieties, enzyme substrates and color reagents as described above. Thediagnostic kit can, alternatively, be an immunoblot kit generallycomprising the components and reagents described herein. The particularreagents and other components included in the diagnostic kits of thepresent invention can be selected from those available in the art inaccord with the specific diagnostic method practiced in the kit. Suchkits can be used to detect the binding of the antibody with hGC-1 andhGC-1 receptor, or a fragment thereof, in tissue and fluid samples froma patient.

One skilled in the art will be able to correlate the levels of hGC-1antigen detected using the methods disclosed herein with a particularstage of the cancer, thus utilizing the detection method for prognosticpurposes. The prognostic evaluation can determine what type ofanti-cancer therapy to employ at different stages of cancer depending onthe amounts of hGC-1 antigen detected in the patient's sample.

Methods

Diagnostic Methods

One method of the present invention is using the nucleic acid of thepresent invention as a marker for the early stages of myeloid lineagedevelopment in hematopoiesis. The method can comprise identifying thepresence of the nucleic acid in a sample of cells. The presence of thenucleic acid can signify the cells tested are of myeloid lineage.

We have shown that hGC-1 expression was limited to early granulocyticprecursor cells in a two phase liquid culture system. In that study,hGC-1 expression in the multipotent prepromyelocytic cell line HL-60 wasobserved only after induction of granulocytic differentiation, notmonocytic differentiation. 32D is a mouse myeloid progenitor cell line,which can differentiate into granulocytes in response to G-CSF. We foundthat expression of mGC-1 (mouse GC-1) was detected on day 7 when 32Dcells were cultured with G-CSF. Therefore, regulation of hGC-1expression is under developmental control during granulocyticdifferentiation.

A method comprising identifying the expression of hGC-1 in a sample ofcells by detecting a nucleic acid specific for the nucleic acid of theinvention is provided. The detection can occur, for example, viadifferential display, northern analysis, or RT-PCR. Expression of hGC-1can indicate a cell is of the normal myeloid lineage of cells.

A method is disclosed comprising identifying a patient at increased riskfor cancer by detecting in the patient a mutation in the gene of theinvention (or identifying a mutation in the hGC-1 gene to determinewhether the patient is at risk for cancer), wherein the presence of amutation indicates increased risk when compared with the hGC-1 level ofa healthy subject, wherein a decrease in the hGC-1 in the patientindicates increased risk. The cancer can be myeloma, B-cell leukemia orprostate cancer; specifically, it can be prostate cancer. The mutationmay be in exon 5 of the gene and may be a deletion. In prostate cancer,it is believed that the mutation, which may be a deletion, may be foundin exon 5.

In a method of the invention, a mutant hGC-1 polypeptide identified isassociated with cancer. Having provided the hGC1 gene and protein, andhaving shown a connection between mutations and disease, the inventionprovides a method for identifying any specific cancer in which hGC1 mayplay a role.

The subject having an increased susceptibility for developing cancer isidentified by detecting a mutated hGC-1 nucleic acid in the subject. Themutated hGC-1 nucleic acid may comprise a missense mutation, that is, amutation that changes a codon specific for one amino acid to a codonspecific for another amino acid. The hGC-1 nucleic acid having asequence associated with cancer may also comprise a nucleic acidsequence having an insertion mutation, where one or more nucleotides areinserted into the wild-type sequence. The mutated hGC-1 nucleic acid maycomprise a deletion mutation, where one or more nucleotides are deletedfrom the wild-type sequence. Such a deletion or insertion mutation may,for example, result in a frameshift mutation, altering the readingframe. Frameshift mutations typically result in truncated (that is,prematurely terminated) hGC-1 polypeptide.

A deletion or insertion mutation may comprise at least one deletion orinsertion at an amino acid position of the sequence set forth in SEQ IDNO:4.

The mutant hGC-1 nucleic acid may also comprise a nonsense mutation,that is, a mutation that changes a codon specific for an amino acid to achain termination codon. Nonsense mutations result in truncated (thatis, prematurely terminated) hGC-1 polypeptide.

A nonsense mutation may comprise at least one mutation at an amino acidposition of the sequence set forth in SEQ ID NO:4.

The mutated hGC-1 nucleic acid may also comprise a truncation mutation,that is, a mutated hGC-1 nucleic acid which encodes a truncated hGC-1polypeptide. This may occur where, for example, the hGC-1 nucleic acidhas a nonsense mutation.

The mutated hGC-1 nucleic acid may comprise a missense mutation, thatis, the mutation can result in a change in a codon such that the mutatedcodon now encodes a different amino acid. The mutation can result in apolypeptide having a non-conservative substitution at the relevant aminoacid residue. One of ordinary skill will readily understand the conceptof a “non-conservative substitution.” Substitutions such as a chargedamino acid for an uncharged amino acid, or an uncharged amino acid for acharged amino acid, or any amino acid in place of a Cys, or visa versa,or any amino acid in place of a Pro, or visa versa, are well known inthe art to alter the structure and often the function of a protein. Themutation can also result in reduction or elimination of hGC-1 mRNAproduction, incorrect or altered processing of hGC-1 RNA, increasedhGC-1 RNA instability, or other effects on expression of hGC-1 prior totranslation. A mutation, which does not alter the encoded amino acid,can affect RNA production, processing, or function.

A missense mutation may comprise at least one mutation at an amino acidposition of the sequence set forth in SEQ ID NO:4.

A non-conservative substitution may comprise at least one substitutionat an amino acid position of the sequence set forth in SEQ ID NO:4.

The hGC-1 nucleic acid having a sequence associated with cancer encodesa mutant hGC-1 polypeptide.

For example, the mutant hGC-1 polypeptide having a sequence associatedwith cancer can comprise at least one mutation at an amino acid positionof the sequence set forth in SEQ ID NO:4. The hGC-1 polypeptide cancomprise at least one mutation at an amino acid position of the sequenceset forth in SEQ ID NO:4.

For example, the hGC-1 polypeptide acid having a sequence associatedwith cancer may comprise at least one mutation at an amino acid positionof the sequence set forth in SEQ ID NO:4.

The hGC-1 polypeptide having a sequence associated with cancer can havea non-conservative amino acid substitution of at least one amino acidresidue of a hGC-1 having the amino acid sequence set forth in SEQ IDNO:4.

The mutated hGC-1 nucleic acid and mutant polypeptide that is detectedcan be from any cause. For example, mutant hGC-1 nucleic acid can be theresult of a familial mutation or sporadic mutation.

Also provided is a method of diagnosing cancer comprising detecting amutation of the hGC-1 gene in suspected cancer cells from a subject. Themutation can be in exon 5.

A method of detecting antibodies that bind to hGC-1 in a biologicalsample comprising the steps of: a) contacting the polypeptide of theinvention with the biological sample suspected of containing thepolypeptide antibodies under conditions that allow for formation of anantibody-antigen complex; and b) detecting the antibody-antigen complex,whereby the presence of the complex indicates the presence of antibodiesthat bind to hGC-1 is disclosed.

A method of detecting hGC-1 or an antigenic fragment thereof in a samplecomprising a) contacting the sample with an antibody which selectivelybinds with the polypeptide of the invention and b) detecting binding ofthe antibody and antigen, whereby the presence of the complex indicatesthe presence of antibodies that bind to hGC-1 is also disclosed. Theantibody may be a purified antibody or fragment thereof, whichspecifically binds to the polypeptide encoded by a nucleic acid of theinvention.

The invention provides a method for detecting the presence of hGC-1antibodies comprising a) binding an hGC-1 polypeptide to a substrate, b)contacting the bound polypeptide with a sample, c) adding secondaryantibodies which bind with the hGC-1 antibodies and which are labeled orbound with a detectable moeity, and d) visualizing the secondaryantibody as well.

A method of detecting a mutant hGC-1 gene comprising a) contacting thesample with an antibody which selectively binds with a mutant hGC-1 andb) detecting binding of the antibody and antigen, whereby the presenceof the complex indicates the presence of antibodies that bind to amutant hGC-1 is provided. The mutant hGC-1 may be associated withdisease such as cancer. The cancer can be myeloma, B-cell leukemia orprostate cancer, specifically prostate cancer.

Therapeutic Methods

A method of delivering a normal hGC-1 gene to cells in a cancer patientor repairing the abnormal hGC-1 gene as a preventative for patients atrisk for cancer or as a treatment for existing cancer is within thescope of the invention. The cancer may be, for example, myeloma, B-cellleukemia, or prostate cancer.

Further provided is a method of treating cancer, comprisingadministering to affected cells of a subject a therapeutically effectiveamount of a non-mutant copy of the gene of the invention, fragment ofthe gene, nucleic acid of the invention, or polypeptide of theinvention. The cancer can be myeloma, B-cell leukemia or prostatecancer. Specifically, the cancer can be prostate cancer.

Additionally, a method of preventing cancer in a subject comprisingadministering a preventative amount of the hGC-1 gene, fragment of thegene, or nucleic acid of the invention to cells of the subject having amutant copy of the gene is provided. The subject may not produce thenormal polypeptide, produce too little of the normal polypeptide, orproduce a mutant of the normal polypeptide.

Still further provided is a method of preventing cancer in a subjectcomprising administering a preventative amount of a polypeptide of theinvention (such as a polypeptide encoded by SEQ ID NO:1; SEQ ID NO:2;SEQ ID NO:3; a sequence complementary to SEQ ID NO:1, SEQ ID NO:2 or SEQID NO:3; or a sequence of at least 10 contiguous nucleotides specificfor SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or a sequence complementary toSEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3) to a subject. The subject maynot produce the normal polypeptide, produce too little of the normalpolypeptide, or produce a mutant of the normal polypeptide.

Nucleic Acid Delivery

In the method described herein which includes the introduction ofexogenous DNA into the cells of a subject (i.e., gene transduction ortransfection), the nucleic acids of the present invention can be in theform of naked DNA or the nucleic acids can be in a vector for deliveringthe nucleic acids to the cells for expression of the nucleic acid insidethe cell. For example, hGC-1 biological activity can be stimulated (orcorrect activity provided) in a subject by administering to the subjecta nucleic acid encoding hGC-1, using any method known for nucleic aciddelivery into the cells of a subject. The hGC-1 nucleic acid is taken upby the cells of the subject and directs expression of the encoded hGC-1in those cells that have taken up the nucleic acid. The vector can be acommercially available preparation, such as an adenovirus vector(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of thenucleic acid or vector to cells can be via a variety of mechanisms. Asone example, delivery can be via a liposome, using commerciallyavailable liposome preparations such as LIPOFECTIN®, LIPOFECTAMINE®(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT® (Qiagen, Inc. Hilden,Germany) and TRANSFECTAM® (Promega Biotec, Inc., Madison, Wis.), as wellas other liposomes developed according to procedures standard in theart. In addition, the nucleic acid or vector of this invention can bedelivered in vivo by electroporation, the technology for which isavailable from Genetronics, Inc. (San Diego, Calif.) as well as by meansof a SONOPORATION® machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells a nucleic acid that encodes a hGC-1 polypeptide. Theexact method of introducing the altered nucleic acid into mammaliancells is, of course, not limited to the use of retroviral vectors. Othertechniques are widely available for this procedure including the use ofadenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994),adeno-associated viral (AAV) vectors (Goodman et al., Blood84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al.,Exper. Hematol. 24:738-747, 1996). Physical transduction techniques canalso be used, such as liposome delivery and receptor-mediated and otherendocytosis mechanisms (see, for example, Schwartzenberger et al., Blood87:472-478, 1996). The present invention can be used in conjunction withany of these or other commonly used gene transfer methods.

In a particular example, to deliver a hGC-1 nucleic acid to the cells ofa human subject in an adenovirus vector, the dosage can range from about107 to 109 plaque forming unit (pfu) per injection but can be as high as1012 pfu per injection (for disclosure of exemplary, non-limiting,dosage ranges, please see Crystal, Hum. Gene Ther. 8:985-1001, 1997;Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997, which are herebyincorporated by reference in their entireties, and specifically for theteaching of gene therapy dosing). Ideally, a subject will receive asingle injection. If additional injections are necessary, they can berepeated at three to six month intervals for an indefinite period and/oruntil the efficacy of the treatment has been established.

For in vivo administration, the cells can be in a subject and thenucleic acid can be administered in a pharmaceutically acceptablecarrier. The subject can be any animal in which it is desirable toselectively express a nucleic acid in a cell. The animal of the presentinvention may be any animal in which selective expression of a nucleicacid in a cell can be carried out according to the methods describedherein.

hGC-1 could be used to modulate hGC-1 activity in vitro in the contextof gene therapy such as for the treatment of cancer. For example, cellscan be collected from the patient and treated ex vivo with hGC-1, washedto remove the hGC-1, then readministered to the patient. This approachoffers the advantage of reducing any side-effects of administration ofhGC-1 in vivo.

The nucleic acid and the nucleic acid delivery vehicles of thisinvention, (e.g., viruses; liposomes, plasmids, vectors) can be in apharmaceutically acceptable carrier for in vivo administration to asubject. The carrier would naturally be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart.

The nucleic acid or vehicle may be administered orally, parenterally(e.g., intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like. Theexact amount of the nucleic acid or vector required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the severity or mechanism of any disorderbeing treated, the particular nucleic acid or vehicle used, its mode ofadministration and the like.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795. For additional discussion of suitableformulations and various routes of administration of therapeuticcompounds, see, e.g., Remington: The Science and Practice of Pharmacy(19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.

Contemplated in this invention is a method of treating or preventingcancer in a subject by administering hGC-1, and, optionally, apharmaceutically acceptable carrier. Alternatively, an active fragmentof hGC-1, or a hGC-1 encoding nucleic acid in an expressible construct,can be administered. For any of the methods described herein for usinghGC-1 or hGC-1 fragments, nucleic acids encoding hGC-1 or its fragmentscan be administered.

The subject which can be treated by this method can be any animal. Theanimal of the present invention can be a human. In addition, non-humananimals which can be treated by the methods of this invention caninclude, but are not limited to, cats, dogs, birds, horses, cows, goats,sheep, guinea pigs, hamsters, gerbils and rabbits.

Optimal dosages used will vary according to the individual being treatedand the hGC-1 being used. The amount of hGC-1 will also vary amongindividuals on the basis of age, size, weight, condition, etc. Oneskilled in the art will realize that dosages are best optimized by thepracticing physician and methods for determining dose amounts andregimens and preparing dosage forms are described, for example, inRemington's Pharmaceutical Sciences. For example, suitable doses anddosage regimens can be determined by comparison to agents presently usedin the treatment or prevention of hGC-1 related disorders. The optimaldosage is the amount of hGC-1 which results in treatment or preventionof cancer, in the absence of significant side effects.

Typically, the hGC-1 of this invention can be administered orally orparenterally in a dosage range of about 0.1 micrograms/kg to about 100mg/kg or about 0.1 mg/kg to about 10 mg/kg depending on the clinicalresponse that is to be obtained. Administration of hGC-1 can be stoppedcompletely following a prolonged remission or stabilization of diseasesigns and symptoms and readministered following a worsening of eitherthe signs or symptoms of the disease, or following a significant changein status, as determined by routine follow-up studies well known to aclinician in this field. Administration may be by any method whichretains desired action.

The efficacy of administration of a particular dose of hGC-1 in treatinga cancer, or a hGC-1 related disorder, as described herein can bedetermined by evaluating the particular aspects of the medical history,the signs, symptoms and objective laboratory tests that have adocumented utility in evaluating pathophysiological activity of theparticular hGC-1 associated disorder being treated. These signs,symptoms and objective laboratory tests will vary depending on theparticular disorder being treated, as will be well known to anyclinician in this field. For example, if, based on a comparison with anappropriate control group and knowledge of the normal progression of thedisorder in the general population or the particular individual, 1) asubject's frequency or severity of recurrences is shown to be improved;2) the progression of the disease or disorder is shown to be stabilized;or 3) the need for use of other medications is lessened, then aparticular treatment can be considered efficacious.

In a particular example, in using the hGC-1 of the present invention totreat cancer, clinical parameters and symptoms which can be monitoredfor efficacy can include reduction in tumor size or full remission ofthe disease.

Once it is established that disease activity is significantly improvedor stabilized by a particular hGC-1 treatment, specific signs, symptomsand laboratory tests can be evaluated in accordance with a reduced ordiscontinued treatment schedule. If a disease activity recurs, based onstandard methods of evaluation of the particular signs, symptoms andobjective laboratory tests as described herein, hGC-1 treatment can bereinitiated.

Additionally, the efficacy of administration of a particular dose of apeptide ligand in preventing an hGC-1 associated disorder in a subjectnot known to have an hGC-1 associated disorder, but known to be at riskof developing an hGC-1 associated disorder, can be determined byevaluating standard signs, symptoms and objective laboratory tests,known to one of skill in the art, over time. This time interval may belong (i.e., years/decades). The determination of who would be at riskfor the development of an hGC-1 associated disorder would be made basedon current knowledge of the known risk factors for a particular disorderfamiliar to clinicians and researchers in this field, such as aparticularly strong family history of a disorder or exposure to oracquisition of factors or conditions which are likely to lead todevelopment of an hGC-1 associated disorder.

Methods of administration can be, for example, oral, sublingual,mucosal, inhaled, absorbed, or by injection. It is also noted that notall methods of administering the present hGC-1 polypeptides or nucleicacids require a pharmaceutically acceptable carrier.

In the present invention, the hGC-1, hGC-1 antibody, or active fragmentcan be orally or parenterally administered in a carrier pharmaceuticallyacceptable to human subjects. Suitable carriers for oral or inhaledadministration of hGC-1 can include one or more of the carrierspharmaceutically acceptable to human subjects. Suitable carriers fororal administration of hGC-1 include one or more substances which mayalso act as a flavoring agents, lubricants, suspending agents, or asprotectants. Suitable solid carriers include calcium phosphate, calciumcarbonate, magnesium stearate, sugars, starch, gelatin, cellulose,carboxypolymethylene, or cyclodextrans. Suitable liquid carriers may bewater, pyrogen free saline, pharmaceutically accepted oils, or a mixtureof any of these. The liquid can also contain other suitablepharmaceutical addition such as buffers, preservatives, flavoringagents, viscosity or osmo-regulators, stabilizers or suspending agents.Examples of suitable liquid carriers include water with or withoutvarious additives, including carboxypolymethylene as a pH-regulated gel.The hGC-1 may be contained in enteric coated capsules that release thepolypeptide into the intestine to avoid gastric breakdown. Forparenteral administration, a sterile solution or suspension is preparedin saline that may contain additives, such as ethyl oleate or isopropylmyristate, and can be injected for example, into subcutaneous orintramuscular tissues, as well as intravenously.

The invention also contemplates a method of reducing hGC1 activity in asubject by administering an antibody to hGC-1 and a pharmaceuticallyacceptable carrier. Alternatively, an antibody to a functional region ofhGC-1 could be administered.

The “sample” of this invention can be from any organism and can be, butis not limited to, peripheral blood, plasma, urine, saliva, gastricsecretion, feces, bone marrow specimens, primary tumors, embedded tissuesections, frozen tissue sections, cell preparations, cytologicalpreparations, exfoliate samples (e.g., sputum), fine needle aspirations,amnion cells, fresh tissue, dry tissue, and cultured cells or tissue. Itis further contemplated that the biological sample of this invention canalso be whole cells or cell organelles (e.g., nuclei). The sample can beunfixed or fixed according to standard protocols widely available in theart and can also be embedded in a suitable medium for preparation of thesample. For example, the sample can be embedded in paraffin or othersuitable medium (e.g., epoxy or acrylamide) to facilitate preparation ofthe biological specimen for the detection methods of this invention.

The term “antibody” is used herein in a broad sense and includes intactimmunoglobulin molecules and fragments or polymers of thoseimmunoglobulin molecules, so long as they exhibit any of the desiredproperties described herein. Antibodies are typically proteins whichexhibit binding specificity to a specific antigen. Native antibodies areusually heterotetrameric glycoproteins, composed of two light (L) chainsand two heavy (H) chains. The heavy and light chains are typicallyidentical, but not necessarily so. Typically, each light chain is linkedto a heavy chain by one or more covalent disulfide bond, while thenumber of disulfide linkages varies between the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain alsotypically has regularly spaced intrachain disulfide bridges. Each heavychain typically has at one end a variable domain (V(H)) followed by anumber of constant domains. Each light chain typically has a variabledomain at one end (V(L)) and a constant domain at its other end; theconstant domain of the light chain is typically aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis typically aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains. The light chains ofantibodies from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains. Depending on the aminoacid sequence of the constant domain of their heavy chains,immunoglobulins can typically be assigned to different classes. Thereare approximately five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term “variable” is used herein to describe certain portions of thevariable domains which differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a -sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the -sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest” NationalInstitutes of Health, Bethesda, Md. (1987)). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainmay be identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) may be identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired antagonistic activity (See, U.S. Pat.No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

As used herein, “antigen” when used in the detection context generallymeans detecting the antigen, specifically hGC-1, or a fragment thereof.The antigens of this invention can also be used to detect antibodies tohGC-1, or fragments thereof.

One example of the method of detecting the antigen is performed bycontacting a fluid or tissue sample from the patient with an amount of aan antibody, possibly purified, reactive with the antigen, cellscontaining the antigen, or fragments of the antigen, and detecting thereaction of the antibody with the antigen. The fluid sample of thismethod can comprise any body fluid which would contain the antigen or acell containing the antigen, such as blood, plasma, serum, saliva andurine, sputum, mucus and the like. An antibody used to detect theantigens of this invention is preferably specifically reactive with theantigen.

In the present invention, the step of detecting the binding of theantibody with the antigen can be further aided, in appropriateinstances, by the use of a secondary antibody or other ligand which isreactive, either specifically with a different epitope ornonspecifically with the ligand or reacted antibody. The antibody can belabeled with a detectable marker.

Enzyme immunoassays such as immunofluorescence assays (IFA), enzymelinked immunosorbent assays (ELISA) and immunoblotting can be readilyadapted to accomplish the detection of the antigen. An ELISA methodeffective for the detection of the antigen can, for example, be asfollows: (1) bind the antibody to a substrate; (2) contact the boundantibody with a fluid or tissue sample containing the antigen; (3)contact the above with a secondary antibody bound to a detectable moiety(e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme);(4) contact the above with the substrate for the enzyme; (5) contact theabove with a color reagent; (6) observe color change. Other assays fordetecting the binding of an antibody to an antigen can be used.

The nucleic acids of this invention can be detected with a probe capableof hybridizing to the nucleic acid of a cell or a sample. This probe canbe a nucleic acid comprising the nucleotide sequence of a coding strandor its complementary strand or the nucleotide sequence of a sense strandor antisense strand, or a fragment thereof. The nucleic acid cancomprise the nucleic acid of the hGC-1 gene, or a sequence associatedwith a gene that is associated with the hGC-1 gene, such as the hGC-1receptor gene. Thus, the probe of this invention can be either DNA orRNA and can bind either DNA or RNA, or both, in the biological sample.The probe can be the coding or complementary strand of a complete geneor gene fragment. The nucleotide sequence of the probe can be anysequence having sufficient complementarity to a nucleic acid sequence inthe biological sample to allow for hybridization of the probe to thetarget nucleic acid in the biological sample under a desiredhybridization condition. Ideally, the probe will hybridize only to thenucleic acid target of interest in the sample and will not bindnon-specifically to other nucleic acids in the sample or other regionsof the target nucleic acid in the sample. The hybridization conditionscan be varied according to the degree of stringency desired in thehybridization. For example, if the hybridization conditions are for highstringency, the probe will bind only to the nucleic acid sequences inthe sample with which it has a very high degree of complementarity. Lowstringency hybridization conditions will allow for hybridization of theprobe to nucleic acid sequences in the sample which have somecomplementarity but which are not as highly complementary to the probesequence as would be required for hybridization to occur at highstringency. Since sequence divergence can exist between individuals forcancer or tumor-related genes, one skilled in the art can take thesepopulation differences into account when optimizing hybridizationconditions. The hybridization conditions will therefore vary dependingon the biological sample, probe type and target. An artisan will knowhow to optimize hybridization conditions for a particular application ofthe present method.

The nucleic acid probes of this invention can be modified nucleic acids.These modified nucleotides are well known in the art and include, butare not limited to, thio-modified deoxynucleotide triphosphates andborano-modified deoxynucleotide triphosphates (Eckstein and Gish, Trendsin Biochem. Sci., 14:97-100 (1989) and Porter Nucleic Acids Research,25:1611-1617 (1997)).

The nucleic acid probe can be commercially obtained or can besynthesized according to standard nucleotide synthesizing protocols wellknown in the art. Alternatively, the probe can be produced by isolationand purification of a nucleic acid sequence from biological materialsaccording to methods standard in the art of molecular biology (Sambrooket al. 1989. Molecular Cloning: A Laboratory Manual, 2d Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). The nucleic acidprobe can be amplified according to well-known procedure foramplification of nucleic acid (e.g., polymerase chain reaction).Furthermore, the probe of this invention can be linked to any of thedetectable moieties of this invention by protocols standard in the art.

The detectable moieties to which the nucleic acid probe of thisinvention can be linked to include, but are not limited to, a hapten,biotin, digoxigenin, fluorescein isothiocyanate (FITC), dinitrophenyl,amino methyl coumarin acetic acid, acetylaminofluorene andmercury-sulfhydryl-ligand complexes, as well as any other molecule orcompound which can be linked to a probe and detected either directly orindirectly according to the methods described herein. One skilled in theart will, therefore, appreciate that a probe, such as a nucleic acidprobe or an antibody, can be labeled with a detectable moiety that canbe directly detected, such as a flurorochrome or a dye, such as achromogenic dye, and the use of secondary reagents to detect the probeis not strictly required.

It is further contemplated that the present invention also includesmethods for oligonucleotide hybridization wherein the hybridizedoligonucleotide is used as a primer for an enzyme catalyzed elongationreaction such as in situ PCR and primed in situ labeling reactions,whereby haptenized nucleotides are incorporated in situ. Additionallyincluded are methods for in situ hybridization, employing syntheticpeptide nucleic acid (PNA) oligonucleotide probes (Nielsen et al., 1991.“Sequence-selective recognition of DNA by strand displacement with athymine-substituted polyamide.” Science 254:1497-1500; Egholm et al.,1993. “PNA hybridizes to complementary oligonucleotides obeying theWatson-Crick hydrogen bonding rules.” Nature 365:566-568).

The levels of protein in this invention can be detected by ELISA, FIA,immunoblotting or any other immunodetection method. These methods can becombined with histochemical or microscopic analysis to determine thelevels of proteins in samples.

By “active hGC-1 gene product” is meant a product of the hGC-1 genewhich can exert a biological function associated with the hGC-1 gene. Anactive hGC-1 gene product may act as a transcriptional activator toelicit downstream effects. The hGC-1 gene product may also function fromoutside of the cell either as a ligand which could bind to its cellsurface receptor and participate in signal transduction, or it mayfunction through the interaction with other secreted proteins andextracellular matrix molecules outside of cells. Any effect associatedwith hGC-1 associated cancers is also contemplated by this invention.

The term “inhibition” is familiar to one skilled in the art and is usedherein to describe any reduction in the activity of the hGC-1 geneproduct. The degree of inhibition does not have to be complete, such ascompletely inhibiting the activity of the hGC-1 gene product, andtherefore comprises any inhibition of the activity of the hGC-1 geneproduct relative to the activity of the hGC-1 gene product in a similarenvironment in the absence of the inhibiting compound.

The “cells” of this invention include any cell type, cancerous ornoncancerous, that may express or be affected by the expression of ahGC-1 gene or the activity of a hGC-1 protein. Examples include, but arenot limited to, prostate cancer cells.

The following Examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods claimedherein are made and evaluated, and are intended to be purely exemplaryof the invention and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in □C or is atambient temperature, and pressure is at or near atmospheric.

One important aim of the study of hematopoiesis is to understand howhematopoietic stem cells undergo lineage restriction (Ogawa, M. (1993)Blood 81, 2844-53). A major advantage of the modified two-phase liquidculture system described herein is that it permits the isolation of arelatively synchronized committed cell population from earlyhematopoietic progenitors to late progenitors (phase I) and fromlineage-committed late progenitor cells (phase II). This system enabledeasy manipulation of the culture conditions and isolation ofhematopoietic cells at various stages. Other systems involving theexpansion of hematopoietic stem cells in liquid cultures of humanprogenitor cells have been described (Warren, M. K., Rose, W. L., Beall,L. D. & Cone, J. (1995) Stem Cells 13, 167-74). There are some inherentlimitations of the system used in the present study, including the useof conditioned medium and fetal calf serum. However, these were largelycontrolled for by explicitly comparing cultures grown in the presence ofone lineage-specific cytokine (e.g., EPO) and not the other two (e.g.,G-CSF or TPO), with other culture conditions remaining constant. Cellswere further enriched by using FACS with lineage-restrictive cellmarkers (FIG. 1). In this manner, and given the fact that cultureconditions prior to the application of G-CSF and EPO were identical, theindividual cytokines became the dominant parameter in determining thesubsequent differential gene expression profiles in the modifiedtwo-phase liquid culture system.

Differential display technology was used to analyze differential geneexpression among early precursors of erythroid, myeloid andmegakaryocytic lineage cells, expecting to find novel lineage-specificgenes or markers and, thus, clues to possible novel functions of knowngenes in hematopoiesis. Others have applied this approach to identifydifferentially expressed genes in very immature (CD34+/CD38− vs.CD34+/CD38+) progenitors (Graf, L. & Torok-Storb, B. (1995) Blood 86,548-56) or to compare gene expansion profiles in normal CD34+ cells withthose in clones derived from acute myeloid leukemia patients (Bond, H.M., Bonelli, P., Mesuraca, M., Agosti, V., Masone, C., Cuomo, C.,Nistico, A., Tassone, P., Tuccillo, F., Cecco, L., Iacopino, L.,Barbieri, V., Cerra, M., Costanzo, F. S., Morrone, G. & Venuta, S.(1998) Stem Cells 16, 13643). The current Examples, below, used verystringent criteria to define a gene as differentially expressed,including a greater than three-fold increased expression of the gene ofinterest in one lineage in two independent experiments and subsequentconfirmation by reverse dot blots and northern blots.

Herein presented is the isolation and characterization of one gene soidentified: the human olfactomedin-like gene hGC-1. hGC-1 appeared to bea useful marker for the early stage of myeloid lineage development,showing strong lineage- and differentiation stage-specific expression.In the modified two-phase liquid culture system, hGC-1 showed anexpression pattern limited to early precursors of the granulocyticlineage, not erythroid or megakaryocytic precursor cells.

As shown, hGC-1 expression in the multipotent prepromyelocytic cell lineHL-60 was observed only after induction of granulocytic differentiation,not monocytic differentiation. Because of the characterization andkinetics of hGC-1 expression, we hypothesize that hGC-1 is underdevelopmental controls during granulocytic differentiation. Our resultsfrom two-phase liquid culture systems showed that hGC-1 was stronglyexpressed in all three lineages during the first day of transition fromphase I to phase II. The reason for this biphasic expression patternremains unknown. Further investigation of the hGC-1 promoter and itsgenomic structure will allow this to be determined and may provide ameans of investigating the regulation of lineage-specific anddifferentiation-related gene expression.

The diverse roles of the extracellular matrix are reflected in itshighly complicated structure; the number of known matrix componentsincreases continually. Yet the mechanisms of extracellular matrixassembly and how they influence cell behavior are only just beginning tobe understood. It is now widely accepted that many functions of cellsand tissues are dynamically regulated by the extracellular matrix. Inaddition to its conventional role in providing a scaffold for buildingtissues, the extracellular matrix acts as a directional highway forcellular movement and provides cells with instructions promotingsurvival, proliferation and differentiation.

hGC-1 and other olfactomedin-related proteins are secreted glycoproteinswith a conserved C-terminal motif. This family of proteins is named fortheir amino acid sequence similarity to olfactomedin, an extracellularmatrix protein of the bullfrog olfactory epithelium (Snyder, D. A.,Rivers, A. M., Yokoe, H., Menco, B. P. & Anholt, R. R. (1991)Biochemistry 30, 9143-53; Bal, R. S. & Anholt, R. R. (1993) Biochemistry32, 1047-53; Yokoe, H. & Anholt, R. R. (1993) Proc Natl Acad Sci USA 90,4655-9). The glycoprotein olfactomedin is specifically expressed in theolfactory neuroepithelium, forming homopolymers held together bydisulfide bonds and carbohydrate interactions. On the basis of itssequence and predicted structure, olfactomedin may function in themaintenance, growth, or differentiation of olfactory cilia. hGC-1 shareshomology with full-length olfactomedin, not only its C-terminal region,and has a very similar secondary structure. However, olfactomedin andhGC-1 have totally different tissue distributions. Olfactomedin ismainly expressed in the brain and nervous system; hGC-1 is onlyexpressed in the digestive system, prostate and bone marrow.Interestingly, another olfactomedin-related glycoprotein, Noelin-1, alsohas a restricted tissue distribution. Noelin-1 is confined to the dorsalneural tube, the ontogenetic precursor of the neural crest, and has animportant role in making the neural tube competent to form neural crest.These data suggest that hGC-1 has a causative role in the development ofthe organs and tissues in which it is expressed.

So far, several members of the family of human olfactomedin-relatedproteins have been cloned; their functions are still being deciphered.The first link between an olfactomedin-related protein and human diseasecame with the discovery of the TIGR protein (Nguyen, T. D., Chen, P.,Huang, W. D., Chen, H., Johnson, D. & Polansky, J. R. (1998) J Biol Chem273, 6341-50); a genetic defect in this molecule may cause juvenile openangle glaucoma (Stone, E. M., Fingert, J. H., Alward, W. L., Nguyen, T.D., Polansky, J. R., Sunden, S. L., Nishimura, D., Clark, A. F.,Nystuen, A., Nichols, B. E., Mackey, D. A., Ritch, R., Kalenak, J. W.,Craven, E. R. & Sheffield, V. C. (1997) Science 275, 668-70). A possibleinvolvement of TIGR in glucocorticoid-induced glaucoma is alsospeculated, in which molecular interactions between TIGR and otherextracellular matrix proteins of the trabecular meshwork may influencehumoral outflow. A recombinant C-terminal truncated TIGR cannot exit thecell, and accumulation of this mutated form inside the cell reducessecretion of the endogenous form (Caballero, M., Rowlette, L. L. &Borras, T. (2000) Biochim Biophys Acta 1502, 447-60).

Additionally, a homologous olfactomedin domain has been found in theN-terminal extracellular region of CIRL (Krasnoperov, V. G., Bittner, M.A., Beavis, R., Kuang, Y., Salnikow, K. V., Chepurny, O. G., Little, A.R., Plotnikov, A. N., Wu, D., Holz, R. W. & Petrenko, A. G. (1997)Neuron 18, 925-37; Ichtchenko, K., Bittner, M. A., Krasnoperov, V.,Little, A. R., Chepurny, O., Holz, R. W. & Petrenko, A. G. (1999) J BiolChem 274, 5491-8), a G-protein-coupled receptor containing domainscharacteristic of cell adhesion proteins. These data suggest that theC-terminal olfactomedin domain may have an important role in secretionof hGC-1 and in extracellular protein signal transduction.

In addition to their common structural domain, olfactomedin familymembers share the trait of having tightly restricted tissue-specificexpression, with different family members being expressed in distinctsets of tissues. These features point to possible involvement ofolfactomedin-like proteins in tissue-specific extracellular regulationof differentiation pathways. The current characterization of the humanolfactomedin-like gene, hGC-1, may contribute to a better understandingof the structure and possible functions of this superfamily.

In summary, a modified method for the liquid culture of humanhematopoietic stem cells to enrich for lineage-expandedprogenitor/precursor cells was developed RNA derived from these cellswas used to clone gene products preferentially or exclusively expressedin early erythroid, myeloid, or megakaryocytic cells. One of these,hGC-1, is primarily expressed as a major extracellular glycoproteinduring granulopoiesis. Characterization of this gene and its productssuggests that hGC-1 may be an extracellular matrix effector of myeloiddifferentiation.

Methods and Materials

Cell Culture and Cell Line

Informed consent was obtained from all blood donors. Peripheral bloodmononuclear cells (pbmcs) obtained from normal human blood donors wereisolated by centrifugation on a gradient of Ficoll-Hypaque (OrganonTeknika Corporation, Durham, N.C.), harvested, and washed twice inDulbecco's PBS. The pbmcs were cultured in a modified two-phase liquidculture system that has been reported previously to permit the growth oferythroid, myeloid and megakaryocytic cells (Liu, W., Wang, M., Tang,D.C., Ding, I. & Rodgers, G. P. (1999) Br. J. Haematol. 105, 459-69). Inbrief, following 7 days of culture (phase I), the nonadherent cells wereharvested, washed and recultured in a phase II medium. Human recombinantEPO (1 U/ml, Ortho Pharmaceutics, Raritan, N.J.), G-CSF (10 ng/ml,Sigma, St. Louis, Mo.), or TPO (10 ng/ml, Gene Technologies Inc.,Rockville, Md.) was added at the beginning of phase II to induceerythroid, myeloid or megakaryocytic lineage differentiation,respectively. The K562, HL-60, MEG-01 and MOLT-4 cell lines werepurchased from ATCC (Manassas, Va.) and cultured in RPMI-1640 mediumsupplemented with 10% FBS in 5% CO₂. HL-60 cells were maintained incontinuous logarithmic growth at densities between 5 and 10×10⁵cells/ml. Experimental cultures were seeded from the parental culture at5×10⁵ cells/ml and induced to differentiate by treatment with 1 μMall-trans retinoic acid (RA) or 10 ng12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. Thedifferentiation of early precursors of these three lineages and HL-60cells was assessed morphologically on cytocentrifuge slides stained withMay-Grunwald and Giemsa stains (Sigma, St. Louis, Mo.). 293 cells (ATCC,Manassas, Va.) were maintained in DMEM containing 10% fetal bovineserum.

Cell Purification by FACS

Monoclonal antibodies (mabs) used for sorting precursors of erythroid,myeloid and megakaryocytic lineages included a fluoresceinisothiocyanate (FITC)-conjugated anti-glycophorin antibody (Immunotech,Coulter, Westbrook, Me.), and anti-CD13 and anti-CD61 antibodiesconjugated to PE (Pharmingen, San Diego, Calif.). On day 5 of phase II,cells were suspended in 0.5 ml of phase II culture medium and incubatedwith the appropriate antibodies at the recommended dilution at 4° C. for30 min. The cells were then washed twice with PBS and resuspended in 1ml of culture medium for fluorescence activated cell sorting (FACS) on aflow cytometer (Epics Elite, Coulter, Hialeah, Fla.).

Differential Display

Cells isolated by FACS were washed twice in PBS, and RNA was extractedusing Trizol Reagent (Molecular Research Center Inc., Cincinnati, Ohio).Differential display was performed with an RNA Image Kit (GenHunter,Nashville, Tenn.). Total RNA was treated with DNase I to removechromosomal DNA contamination with a Messageclean Kit (GenHunter) priorto performing differential display experiments. Reverse transcriptionwas carried out by incubating a 20-μl reaction mixture containing 0.2 μgDNA-free total RNA, 1.6 μl of each dNTP (250 μM), 4.0 μl 5×RT buffer,2.0 μl of one of the three different one-base anchored H-T₁₁M primers (2μM, where M may be G, A, or C) at 65° C. for 5 min, 37° C. for 60 min,and 75° C. for 5 min. After the tubes had been held at 37° C. for 10min, the thermocycler was paused; 1 μl of MMLV reverse transcriptase wasadded to each tube and quickly mixed by finger tapping before continuingincubation. The polymerase chain reaction was carried out with 16arbitrary primers (H-AP1 through H-AP16). In a total volume of 20 μl,the reaction mixture contained 2 μl RT-mixture from the previous step,1.6 μl of each DNTP (25 μM), 2.0 μl 10×PCR buffer, 2.0 μl H-AP primer (2μM), 2 μl H-T₁₁M (2 μM), 0.2 μl [α-³³P]-dATP (2000 Ci/mmole), and 0.2 μlTaq DNA polymerase (Qiagen Inc., Valencia, Calif.). PCR was carried outusing a Thermal Cycler 480 (Perkin Elmer, Norwalk, Conn.) at 94° C. for30 seconds, 40° C. for 2 min, and 72° C. for 30 seconds for 40 cycles,followed by 72° C. for 5 min. The resulting PCR products were subjectedto 6% denaturing polyacrylamide gel electrophoresis (PAGE). The gelswere then dried and exposed to Biomax film (Kodak, Rochester, N.Y.)overnight. Each reaction was performed in duplicate from independent RNAsample preparations. Only the differentially expressed bands that werereproducible on the gel were excised and eluted by boiling the gelslices along with the associated 3M drying paper for 15 min. Elutedproteins were then precipitated with 100% ethanol in the presence of 3 Msodium acetate and 10 mg/ml glycogen as a carrier. Reamplification wasperformed using the same primer set and PCR conditions, except that thedNTP concentrations were 20 μM instead of 2 μM and no isotope was added.The PCR products were run on agarose gels and checked to see if thesizes of reamplified PCR products were consistent with their sizes onthe PAGE gel. The reamplified cDNA probe was extracted from the agarosegel using a Qiaex II gel extraction kit (QIAGEN).

cDNA Cloning and Sequence Analysis

5′-RACE and 3′-RACE were performed using the Marathon™ cDNAAmplification Kit and bone marrow Ready Marathon™ cDNA (Clontech, PaloAlto, Calif.). Gene-specific primers were designed according to theoriginal hGC-1 partial cDNA sequence derived from differential display.The PCR products of 5′-RACE and 3′-RACE were purified from a 0.8%agarose gel and cloned into the TA vector. The full-length cDNA sequencewas obtained by overlapping sequences derived from 5′RACE, 3′RACE andthe original differential display sequence. GCG (Genetics ComputerGroup, Madison, Wis.) Version 8 was used to carry out homology searchesof GenBank, EBI, SwissProt, and EST databases.

Fluorescence in situ Hybridization and Chromosomal Mapping of hGC-1

Metaphase chromosome spreads were prepared from human peripheral bloodlymphocytes. Standard FISH protocols were followed (Pack, S. D., Zbar,B., Pak, E., Ault, D. O., Humphrey, J. S., Pham, T., Hurley, K., Weil,R. J., Park, W. S., Kuzmin, I., Stolle, C., Glenn, G., Liotta, L. A.,Lerman, M. I., Klausner, R. D., Linehan, W. M. & Zhuang, Z. (1999)Cancer Res 59, 5560-4). BAC DNA containing hGC-1 was labeled bynick-translation (Roche, Indianapolis, Ind.) with digoxigenin-11-dUTP,and ethanol-precipitated in the presence of a 50-fold excess of herringsperm DNA and a 50-fold excess of C_(o)t-1 human DNA. Biotinylatedprobes were detected with FITC-conjugated avidin (Vector) anddigoxigenin-labeled probes and a rhodamine-conjugated anti-digoxigeninantibody (Roche, Indianapolis, Ind.). Chromosomes were counterstainedwith 4,6-diamidino-2-phenylindole (DAPI). Images were captured with aZeiss epifluorescence microscope equipped with a thermoelectricallycooled CCD camera (Photometrics CH250). As a second independent mappingmethod, PCR was also performed to detect hGC-1 sequences in theGenebridge 4 Radiation Hybrid Screening Panel (Research Genetics, Inc.)using a set of primers designed based on the cDNA sequence of hGC-1:5′-CTGATGGCAG TGACAAAGTGC-3′ (SEQ ID NO:5), 5′-TGTAGTGTATGTGGTCGTTC-3′(SEQ ID NO:6). PCR was carried out in a 20-μl reaction volume for 35cycles. Each cycle consisted of denaturation at 94° C. for 30 s andannealing at 68° C. for 3 min, and 10 μl of each reaction was analyzedby electrophoresis through an 0.9% agarose gel in TAE buffer. The PCRresults of the radiation hybrid panel were mapped for genes relative tothe radiation hybrid map of the human genome (Hudson, T. J., Stein, L.D., Gerety, S. S., Ma, J., Castle, A. B., Silva, J., Slonim, D. K.,Baptista, R., Kruglyak, L., Xu, S. H. & et al. (1995) Science 270,1945-54).

Northern Blotting

Total RNA (10 μg) extracted from glycophorin A+ (erythroid), CD13+(myeloid) and CD61+ (megakaryocytic) cells and four cell lines (K562,HL-60, MEG-01 and MOLT-4 cells) was subjected to electrophoresis ondenaturing formaldehyde gels with 1% agarose. The RNA was thentransferred to a NYTRAN nylon membrane overnight using the Turboblotter™System (Schleicher & Schuell, Keene, N.H.) and exposed to UV light forcross-linking as above. The cDNA probes were made by random primerlabeling with [α-³²P]-dCTP using the Prime-It® RmT Kit (Stratagene, LaJolla, Calif.) and purified on Probe Quant™ G-50 microcolumns as above.Membranes were pre-incubated with ExpressHyb solution (Clontech) at 68°C. for 30 min. and hybridized with a radiolabeled probe at 68° C. forone hour. After removal of the radiolabeled probe, the membrane waswashed first in a solution containing 2×SSC, 0.05% SDS at roomtemperature for 30 min., then washed in 0.1×SSC, 0.1% SDS solution withcontinuous shaking at 50° C. for 40 min. The membrane was exposed toX-ray film at −70° C. overnight. Membranes were stripped between probesby incubating the blots in sterile H₂O containing 0.5% SDS at 95° C. for10 min. Expression of hGC-1 in multiple human tissues was determined byhybridization of cDNA probes to Multiple Tissue Northern (MTN™) Blots(Clontech) according to the hybridization conditions described above.

Reverse Transcriptase (RT)-PCR

Total RNA was prepared from cells by using Trizol reagent, (MolecularResearch Center, INC. Cincinnati, Ohio), and cDNA was prepared from 2 μgof total RNA with oligo(dT) priming using the SuperscriptPreamplification System (Life Technologies, Gaithersburg, Md.). RT-PCRanalyses were performed by using 1/40 of the reverse transcriptionreaction mixture (the amount of cDNA derived from 50 ng of total RNA) asa template to maintain a constant amount of input cDNA for all samplesanalyzed. PCR amplification using AmpliTaq (PE Biosystems, Poster City,Calif.) was carried out so that the reaction was completed within theexponential cycling phase. The PCR conditions were 5 minutes at 94° C.,followed by cycling for 30 seconds at 94° C., 15 seconds at 60° C., and30 seconds at 72° C., followed by elongation for 7 minutes at 72° C.;both hGC-1 and β-actin were amplified for 35 cycles. PCR products (20μl) were fractionated on 2% agarose gels and visualized after ethidiumbromide staining. Because yields of RNA preparations may vary, equalamounts of RNA were used for cDNA preparations. For all samples, cDNAderived from 50 ng of total RNA was amplified, and β-actin messagelevels were assessed. Oligonucleotide PCR primers (shown with finalproduct size) were as follows: β-actin (650 bp)5′-CTGGCCGGGACCTGACTGACTACCTC-3′ (SEQ ID NO:7) and5′-AAACAAATAAAGCCATGCCAA TCTCA-3′ (SEQ ID NO:8); hGC-1 (630 bp),5′-GATTACTCTCCCCAGCATC-3′ (SEQ ID NO:9) and 5′-CTCTTT CACCCTAACTCC-3′(SEQ ID NO:10).

In vitro Translation and N-Glycosylation of hGC-1

To define the biochemical features of hGC-1, in vitro translation andposttranslational processing were carried out. The PCR-amplified hGC-1protein coding region was subcloned into the PCR II TOPO vector(Invitrogen). The resulting PCRII-TOPO-hGC-1 construct was sequenced toconfirm that it had the same sequence as the cDNA. hGC-1 plasmidsPCRII-TOPO-hGC-1 and pcDNA-E-hGC-1-his-V₅ (subcloned as described below)were employed as templates, using a TNT Quick T7 coupledtranscription/translation system (Promega) with [³⁵S]-methionine (15mCi/ml, Amersham Pharmacia Biotech). Labeled hGC-1 was analyzed on 4-12%Bis-Tris gels after incubation with or without canine pancreaticmicrosomal membranes (CPMM) (Promega) to N-glycosylate the protein.

Transfection, Western Blotting and N-Glycanase Analysis of hGC-1

The PCR-amplified hGC-1 coding region was subcloned into thepUni/V5-His-TOPO donor vector (Echo Cloning System, Invitrogen). Theresulting pUni-hGC-1 was sequenced to confirm that it had the samesequence as the cDNA. By mixing the donor pUni-hGC-1 with the acceptorvector pcDNA3.1-E and the Cre recombinase, a fusion plasmidpcDNA3.1-E-hGC-1 was created. This plasmid contained the V5-His epitopetag, and could be used to express the hGC-1 product in vitro and in 293cells. 293 cells were transfected with pcDNA3.1-E-hGC-1 by calciumphosphate coprecipitation. Thirty-six hours after transfection, 293cells in 10-cm culture dishes were harvested by gentle scraping in 1 mlof ice-cold PBS and pelleted by centrifugation at 1200 rpm at 4° C. ThePBS was aspirated and the cell pellet (10⁶ cells) resuspended in 0.5 mlof ice-cold buffer A [which contained 50 mM Tris-Cl (pH 7.4), 150 mMNaCl, 5 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10μg/ml aprotinin, 10 μg/ml leupeptin]. The lysis mixture was rotated 360°for 30 min at 4° C. and then cleared by centrifugation at 12,000×g for10 min at 4° C. Cell lysates from non-transfected andpcDNA-E-hGC-1-his-V₅ transfected 293 cells were treated in the presenceor absence of PNGase (New England BioLabs). Samples containing 10 μg ofprotein were subjected to electrophoresis on 4-12% SDS-PAGE, followed bywestern blotting with a horseradish peroxidase-conjugated anti-V₅ mab(1:5000) (Invitrogen). V₅ immunoreactivity was visualized directly withenhanced chemiluminescence (ECL System, Amersham, Arlington Heights,Ill.) according to the manufacturer's instructions.

Example 1 In vitro Induction of Three Hematopoietic Lineages andEnrichment of Glycophorin A+ (Erythroid), CD13+ (Myeloid) and CD61+(Megakaryocytic) Cells

Peripheral blood mononuclear cells were isolated as described above andincubated in phase I medium for one week. The cells were then collectedand washed, and lineage-specific cytokines were added. For erythroid andmyeloid lineage differentiation, EPO (1 U/ml) and G-CSF (10 ng/ml)respectively were added to the phase II medium (containing 30% FBS) atthe beginning of phase II. For megakaryocytic lineage development, TPO(10 ng/ml) was added to serum-free medium that contained 20% BIT (FIG.1). On day 5, in phase II culture medium, glycophorin A+ (erythroid),CD13+ (myeloid) and CD61+ (megakaryocytic) cells were isolated usingtheir corresponding FITC- or PE-conjugated mabs and FACS (FIG. 1). Theaverage percentage of positive cells in each induced cell population inpresorted samples was 9.4%, 8.6% and 6.2%, respectively. The developmentof the three lineages was monitored morphologically on cytocentrifugeslides (FIG. 1).

Example 2 Differential Display and Determination of the Full-Length cDNASequence

Total RNA was isolated from glycophorin A+, CD13+ and CD61+ cells,reverse-transcribed, and amplified by PCR in the presence of[α-³³P]-dATP. A total of 16 5′-arbitrary primers (H-AP1 through H-AP16)in combination with three 3′ one-base-anchored oligo(dT) primers(H-T₁₁A, H-T₁₁C and H-T₁₁G) were used. The PCR products were displayedon 6% DNA sequence gels and autoradiographed. Each differential laneyielded 75-100 discrete bands, allowing evaluation of more than 10,000RNAs, thought to represent about 50% of the estimated repertoire of15,000-20,000 cellular mRNAs. Bands that showed at least a three-foldincreased expression pattern among the three lineages, and werereproducible in two independent mRNA sample preparations, were elutedand subjected to PCR reamplification. Among the 48 primer sets, 130bands of interest were excised and eluted, of which 112 bands werereamplified successfully.

The cDNA fragment on differential display gels that led to the hGC-1myeloid clones we eventually analyzed is shown in FIG. 2A. 5′-RACE and3′-RACE were performed using a Marathon cDNA amplification kit and bonemarrow Marathon-Ready cDNA (Clontech). The original hGC-1 sequencecomprising 200 bp from the differential display isolate was used todesign gene-specific primers as follows: 5′-RACE:5′-GCACATCACATACACCAGCAAGG-3′ (SEQ ID NO:11). 3′-RACE: 5′-CAGTGCAGTAGTTGGAAACCTTGCTGG-3′ (SEQ ID NO:12). The full-length sequence of hGC-1was obtained by overlapping the 5′-RACE, 3′-RACE and originaldifferential display sequences. FIG. 2B shows the nucleotide sequence ofhGC-1 (SEQ ID NO:2). This sequence was also confirmed by screening thehGC-1 sequence against the database of human expressed sequence tags(dbEST) at the NCBI (http://www.Ncbi.nlm.nih.gov/dbEST). cDNA cloneswith significant homology were identified, obtained from Genome Systemsand sequenced. The human EST clone encoding the full-length hGC-1 cDNAwas deposited by the I.M.A.G.E. consortium (I.M.A.G.E. ID: 457140) anddistributed by Genome Systems. The hGC-1 cDNA is 2849 nucleotides inlength, and its polyadenylation signal is located at nucleotide 2818.

Example 3 Gene Structure and Chromosomal Localization of hGC-1

Using the basic BLAST search, we screened all sequences in the GenBank,EMBL, DDBJ and PDB databases with hGC-1 cDNA and obtained a humangenomic DNA sequence from clone RP11-209J19 on chromosome 13 (Accession:AL390736) with significant homology. Then intron-exon boundaries weredefined by comparing the genomic sequence and the cDNA sequence. Asshown in FIG. 3A, the hGC-1 gene consists of 5 exons spanning over 23kb. The sizes of the exons range from 156 (exon 2, 4) to 801 bp (exon 5,last exon), whereas those of the introns vary from 0.98 (intron 3) to7.4 kb (intron 2).

To identify the chromosomal localization of hGC-1, we performed FISH andradiation hybrid mapping. We performed FISH analysis with full-lengthhGC-1 cDNA as a probe and observed a weak signal on chromosome 13q.Therefore, we performed a labeling FISH analysis with the BAC clonecontaining the hGC-1 gene. We obtained strong signals at chromosomeposition 13q14.3 (FIG. 3B). To confirm this localization, we employedradiation hybrid mapping using the Genebridge 4 radiation hybrid panel.PCR products of the expected size (300 bp) were amplified from 29 out of93 hybrid cell lines. Comparison with the human chromosomal content ofthe hybrids, as determined by the Whitehead Institute/MIT Center forGenome Research, localized the hGC-1 gene to chromosome 13 and placed it2.63 cR from D13S153, locating it on human chromosome 13q14.3 (FIG. 3C).Both Rb and BRCA-2, two important tumor suppressors, are also located on13q14.

Example 4 Expression Patterns in Leukemia Cell Lines and MultipleTissues

To further characterize hGC-1, we examined the expression patterns ofhomologous transcripts in K562, HL-60, MEG-01 and MOLT-4 cell lines(FIG. 4A) and 22 normal human tissues (FIG. 4B) by northern blothybridization. hGC-1 was specifically expressed in the myeloid (CD13+)lineage, but not expressed in K562, HL-60, MEG-01 or MOLT-4 cell lines.The prepromyelocytic cell line HL-60 expresses hGC-1 only when thesecells are induced to differentiate towards granulocytes, but not underconditions of forced monocytic differentiation. The protein sequence ofhGC-1 indicates that it is a member of the olfactomedin-related familyof glycoproteins, which includes olfactomedin, TIGR, NOELIN-2 andlatrophilin-1. Like other olfactomedin-like genes with tissue-restrictedpatterns of expression, hGC-1 is expressed only in prostate, smallintestine, colon, bone marrow, and stomach, and is absent in all othertissues examined. In vitro translation and ex vivo expression showedthat hGC-1 is an N-linked glycoprotein.

Example 5 Kinetics of hGC-1 Expression During Hematopoiesis

Our studies of hGC-1 expression using differential display and northernanalysis indicated that expression of this gene is limited to themyeloid lineage. To gain more insight into the time course of hGC-1expression during myeloid lineage development, we performed the moresensitive RT-PCR assays. Peripheral blood mononuclear cells wereisolated as described above and incubated in phase I medium for oneweek. The cells were then collected and washed, and lineage-specificcytokines were added. For erythroid and myeloid lineage differentiation,EPO (1 U/ml) and G-CSF (10 ng/ml) were respectively added to mediumcontaining 30% FBS at the beginning of phase II. For megakaryocyticlineage development, TPO (10 ng/ml) was added to serum-free mediumcontaining 20% BIT. On days 1, 3, 5, 7, 9, and 11, cells were collected,and hGC-1 expression was determined by RT-PCR (FIG. 5B). Interestingly,on day 1, hGC-1 was expressed in all three lineages. But after that,hGC-1 was expressed only in the myeloid lineage, not in the erythrocyticor megakaryocytic lineages. The promyelocytic HL-60 cell line wasestablished from cells of a patient with AML type M2; these cells can beinduced to differentiate toward cells carrying granulocytic or monocyticmarkers when cultivated with certain physiologic or nonphysiologicinducers. Although hGC-1 expression was not detected beforedifferentiation, it was expressed after RA-induced granulocyticdifferentiation, but was not detected after phorbol ester-inducedmonocytic differentiation (FIG. 5D). These findings confirm the resultsof the kinetics of hGC-1 expression determined in the two-phase culturesystem, and suggest that hGC-1 expression is specific to the myeloidlineage.

Example 6 hGC-1 is an Olfactomedin-Related Glycoprotein

The largest open reading frame of the hGC-1 cDNA predicted a protein of510 amino acids (FIG. 2B). The predicted protein had a signal sequence,but no apparent transmembrane domain. The mature protein, whichconsisted of 490 amino acids, had a calculated molecular weight of55,642, which is in close agreement with the observed size of in vitrotranslated hGC-1 (55 kDa). hGC-1 contained six potential N-linkedglycosylation sites evenly distributed throughout its sequence.Structural analyses of the hGC-1 cDNA sequence demonstrated its proteinto be an extracellular molecule with very high amino-acid sequencesimilarity (65%) to olfactomedin, a glycoprotein found in the olfactoryepithelium of the bullfrog (Yokoe, H. & Anholt, R. R. (1993) Proc NatlAcad Sci USA 90, 4655-9). Olfactomedin was subsequently found to beexpressed throughout the mammalian brain. The TIGR/myocilinolfactomedin-related protein is expressed in the eye and is associatedwith the pathogenesis of glaucoma. The link between TIGR/myocilin andocular hypertension and the fact that several of these proteins areexpressed in various mucus-lined tissues suggests that they function inregulating specific physical properties of the extracellularenvironment. Based on Chou-Fasman analysis, hGC-1 was predicted to havea secondary structure significantly similar to olfactomedin, which has apredominantly α-helical structure at its N-terminal, a mostly β-sheetconfiguration near its C-terminal third, and a region characterized byseveral turns in the center (FIG. 6A). One significant differencebetween these two molecules was that hGC-1 is more hydrophobic thanolfactomedin. The C-terminal region of hGC-1 also had 46% amino acidsimilarity to Noelin-1 (Barembaum, M., Moreno, T. A., LaBonne, C.,Sechrist, J. & Bronner-Fraser, M. (2000) Nat Cell Biol 2, 219-25), asecreted protein that has the ability to prolong neural crestproduction, 49% similarity to the TIGR protein (trabecular-meshworkinducible glucocorticoid response protein), which has been implicated insome glaucomas (Nguyen, T. D., Chen, P., Huang, W. D., Chen, H.,Johnson, D. & Polansky, J. R. (1998) J Biol Chem 273, 6341-50), and 47%similarity to CIRL (calcium-independent receptor of a-latrotoxin), amember of the G-protein-coupled receptor family (Krasnoperov, V. G.,Bittner, M. A., Beavis, R., Kuang, Y., Salnikow, K. V., Chepurny, O. G.,Little, A. R., Plotnikov, A. N., Wu, D., Holz, R. W. & Petrenko, A. G.(1997) Neuron 18, 925-37). These analyses suggested that the C-terminalolfactomedin-like domain is highly conserved among these proteins (FIG.6B).

Example 7 In vitro Translation and N-Glycosylation of hGC-1 Protein

To assess ER translocation and early processing events duringbiosynthesis, hGC-1 was translated in vitro, in either the absence orthe presence of CPMM as an ER and N-glycosylation source, and analyzedby SDS-PAGE. In the absence of CPMM, in vitro translated hGC-1, with orwithout a c-His-V5 tag, migrated as a single band with a molecular massslightly higher than the predicted molecular mass of 53 kDa (FIG. 7A).In the presence of CPMM, the hGC-1 proteins exhibited a slower migrationpattern, suggesting that they may be differentially glycosylated. Thesein vitro studies indicated that hGC-1 could be glycosylated.

Example 8 Western Blotting and N-Glycanase Analysis of hGC-1 Protein

To establish the in vivo expression of hGC-1 as a glycoprotein, celllysate proteins were isolated from 293 cells transfected with thehGC-1-His/V5 construct. Western blots probed with the anti-V5 monoclonalantiserum showed a major protein band with a molecular mass of ˜64 kDa(FIG. 7B, line 3); this size was slightly larger than that of in vitroglycosylated hGC-1 (FIG. 7A, lane 3). After N-glycanase treatment, thesize of hGC-1 decreased to 54 kDa (FIG. 7B, lane 4), equivalent to thatof in vitro translated but unglycosylated hGC-1 (FIG. 7A, lane 2). Thisresult indicated that the same AUG initiation codon was used for hGC-1synthesis both in vivo and in vitro.

Example 9 Molecular Studies on Presence and Expression of hGC-1

Studies were done to determine whether hGC-1 was present in andexpressed in various cell lines and prostate tissues. FISH, PCR, andRT-PCR were conducted as described above or as described in theliterature. The results of these studies are shown in Table 1. Theresults indicate that, while a copy of hGC-1 appears to be present inthe normal and cancer tissues and cells tested (FISH), hGC-1 is notexpressed in prostate cancer or myeloma cell lines, or in prostatecancer tissue or benign prostate hypertrophy (BPH) tissue (RT-PCR).Additionally, the gene was found to be expressed in 2/2 normal tissue,but only in 10/66 prostate cancer on a multi-tumor tissue arraydeveloped by the NCI (Table 2). These results suggest that while thegene may be intact in most of the tissues studied, hGC-1 is more likelynot to be expressed in pre-cancerous or cancer cells.

TABLE 1 Results of detection of hGC-1 in various cells and tissues.Cells Prostate Cell lines Tissues (prostate) Technique epithelial cellsProstate Myeloma Colon BPH Normal Cancer Number tested 1 4 4 4 4 5 7FISH + + +(2) + −(3) +(3) +(5) −(2) N/A (1) N/A (2) N/A (2) PCR (DNA) ++(3) N/A N/A −(3) +(1) −(4) −(1) N/A (1) −(2) N/A (3) N/A (2) RT-PCR(RNA) + − − N/A − +(2) − −(3) + = presence of gene found − = gene notfound N/A = Not available (x) = number of samples for result if not allsamples tested the same

TABLE 2 Detection of hGC-1 Gene in Prostate Multitissues ProstateMultitissues Techniques Normal (2) Tissues (with Prostate Cancer) ISH+(2) +(10/66)

Example 10 Identification of Mouse GC-1 as an Olfactomedin-RelatedGlycoprotein

As described above, cloning of human GC-1 demonstrated that this geneincludes the conserved C-terminal motif that characterizes theolfactomedin-related glycoprotein family of genes. To determine whetherthe C-terminal olfactomedin motif was also conserved in mice, wescreened a mouse expressed sequence tag (EST) database using the hGC-1cDNA sequence. Several mouse cDNA clones with significant homology tohGC-1 were identified. These ESTs were obtained from Incyte Genomics(Palo Alto, Calif.) and subjected to complete DNA sequence analysis. Thelargest of these cDNA clones contained an open reading frame of 1515 bpflanked by 5′- and 3′-untranslated regions and encoded a polypeptide of505 amino acids with a predicted molecular mass of 54 kDa. Comparison ofthe coding sequence of mGC-1 with hGC-1 revealed that these proteinsshare 93% identity at the amino acid level.

Example 11 Gene Structure, Chromosomal Localization, and Expression ofthe Mouse GC-1 Gene

Using the basic BLAST search, we screened all sequences in the genedatabases of Celera Genomics (Rockville, Md.) with the mGC-1 cDNA andidentified a mouse genomic DNA sequence on chromosome 14 (Accession:AL390736) with significant homology. Intron-exon boundaries were definedby comparing the genomic sequence and the cDNA sequence. The mGC-1 genecontains five exons spanning over 23 kb, with sizes ranging from 156 bp(exons 2 and 4) to 801 bp (exon 5); the size of the introns ranges from0.98 (intron 3) to 7.4 kb (intron 2). We localized mGC-1 by FISH(fluorescence in situ hybridization) and radiation hybrid mapping. Alabeling FISH analysis with the BAC clone containing the mGC-1 gene gavestrong signals at chromosome position 14D3. We examined the expressionpattern of mGC-1 in twelve tissues of normal adult mice by Northern blotanalysis. A transcript of approximately 2.4-kb was detected mostintensely in small intestine, and was also expressed in kidney, spleen,stomach, and thymus. To determine the distribution of mGC-1 mRNA duringmurine embryogenesis, in situ hybridization studies were performed ontissue sections from intact mouse embryos on days 8, 9, 10, 11, 12, 13,14, 15, and 16 during development. This analysis revealed that mGC-1 wasnot expressed until day 15. Analysis of E15 and E16 embryos and newbornmice showed that high-level expression of mGC-1 mRNA occurred primarilyin the digestive system. mGC-1 was weakly expressed in the pancreas onday 15, and strongly expressed in the pancreas on day 16. hGC-1 wasexpressed in the digestive tracts of newborn mice. To determine thespecific distribution of mGC-1 within the digestive system, sections ofthe adult mouse digestive tract were subjected to in situ hybridization.As expected, mGC-1 expression was detected in the small intestine andstomach. Interestingly, a very clear demarcation of the hGC-1 signal wasevident in the intestine, where mGC-1 was specifically expressed betweenthe enterocytes lining the villi, but not in the lamina propria or inthe muscularis layers. There was a similar, albeit weaker, pattern ofmGC-1 expression in stomach. mGC-1 expression during 32D celldifferentiation induced by G-CSF. 32D is a murine interleukin 3(IL-3)-dependent myeloblastic cell line that can be induced todifferentiate toward granulocytes by exposure to G-CSF. Although mGC-1was not expressed prior to differentiation, it was expressed on day 7after G-CSF-induced granulocytic differentiation. This result isconsistent with our findings from the two-phase culture system, showingthat hGC-1 expression is specific to the human granulocytic lineage.Thus, these findings suggest that mouse GC-1 expression is also specificto the mouse granulocytic lineage.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. An isolated nucleic acid encoding a protein having the amino acidsequence of SEQ ID NO:
 4. 2. The nucleic acid of claim 1, wherein saidnucleic acid has the sequence of SEQ ID NO:
 1. 3. A vector comprisingthe nucleic acid of claim 1, wherein the vector is suitable forexpressing the nucleic acid.
 4. A vector comprising the nucleic acid ofclaim 2, wherein the vector is suitable for expressing the nucleic acid.5. An isolated cell comprising the vector of claim
 3. 6. An isolatedcell transfected or transformed with the nucleic acid of claim
 1. 7. Thecell of claim 6, wherein the cell is a mammalian, bacterial, yeast, orinsect cell.
 8. An isolated cell comprising the vector of claim
 4. 9. Anisolated cell transfected or transformed with the nucleic acid of claim2.
 10. A kit comprising a packaging, containing: the nucleic acid ofclaim
 1. 11. An isolated nucleic acid of at least 750 nucleotides with95% or greater overall homology to the nucleic acid of SEQ ID NO:
 1. 12.An isolated nucleic acid having a sequence with 96% or greater homologyto the nucleic acid of claim
 11. 13. An isolated nucleic acid having asequence with 97% or greater homology to the nucleic acid of claim 11.14. An isolated nucleic acid having a sequence with 98% or greaterhomology to the nucleic acid of claim
 11. 15. An isolated nucleic acidhaving a sequence with 99% or greater homology to the nucleic acid ofclaim
 11. 16. An isolated nucleic acid of at least 750 nucleotides fullycomplementary to SEQ ID NO:1 or its complement.